Novel and potent adenosine 3',5'-cyclic phosphate phosphodiesterase III inhibitors: thiazolo[4,5-b][1,6]naphthyridin-2-ones.

The transformation of 3-bromo-1,6-naphthyridin-2(1H)-ones 8 to thiazolo[4,5-b][1,6]naphthyridin-2(1H)-ones 12 resulted in a 2-9-fold increase in cAMP phosphodiesterase (PDE) III inhibitory potency. Unlike the secondary binding sites on the cAMP PDE III isozyme which interact with the methyl group of milrinone (2) and CI-930 (4), the site which interacts with the 5-substituents of 1,6-naphthyridin-2(1H)-ones and the 8-substituents of thiazolo[4,5-b][1,6]naphthyridin-2(1H)-ones 12 is able to accommodate a diverse group of substituents which have different steric and electronic requirements.

Novel cAMP PDE III inhibitors: 1,6-naphthyridin-2(1H)-ones.

Two series of medorinone (3) analogs were prepared by modifications at C(2) and C(5). The C(2)-series was prepared from 2-chloro-5-methyl-1,6-naphthyridine (4) by replacement of the chloro group with various nucleophiles. The C(5)-series was prepared from 5-acyl-6-[2-(dimethylamino)-ethenyl]-2(1H)-pyridinone (11), 5-bromo-1,6-naphthyridin-2(1H)-one (17), and 1,3-diketones 19 and 27. 1,6-Naphthyridin-2(1H)-ones are novel inhibitors of cAMP PDE III. Modification of the carbonyl group of 3 or N-methylation at N(1) resulted in a dramatic loss of enzyme activity. Absence of the C(5)-methyl group of medorinone (3) or its shift to C(3) or C(7) also resulted in reduced activity. Substitution at C(3) also diminished activity. However, substitution at C(5) by a wide variety of substituents led to improvement of enzyme activity and several C(5)-substituted analogs were more potent than milrinone.

Relationship between ethanol-induced alterations in fluorescence anisotropy and adenylate cyclase activity.

The effects of butanol, ethanol, and ketamine on adenylate cyclase activity and fluorescence anisotropy were determined in membranes prepared from L6 cells. The experiments were designed to test the hypothesis that the effects of ethanol on adenylate cyclase activity are a consequence of ethanol-induced changes in bulk membrane order. Butanol and ethanol elicited concentration-dependent increases in adenylate cyclase activity and caused decreases in the fluorescence anisotropy of diphenylhexatriene. Butanol was more potent than ethanol in reducing fluorescence anisotropy, and it elicited a greater reduction in fluorescence anisotropy than did ethanol. Butanol was also more potent than ethanol in activating adenylate cyclase, but the highest concentration of butanol used caused a smaller increase in enzyme activity than did the highest concentration of ethanol. When the percent change in adenylate cyclase activity was plotted against the percent change in fluorescence anisotropy at each concentration of alcohol, the increase in isoproterenol-stimulated adenylate cyclase activity per unit change in fluorescence polarization was greater with ethanol than with butanol. Ketamine decreased fluorescence anisotropy but, unlike the alcohols, ketamine caused a decrease in adenylate cyclase activity. A reduction in assay temperature attenuated both the ethanol-induced activation of adenylate cyclase activity and the ethanol-induced reduction in fluorescence anisotropy. Although the data are consistent with the theory that ethanol acts upon a hydrophobic region of the membrane to enhance adenylate cyclase activity, activation of the enzyme does not appear to be a consequence of a decrease in bulk membrane order.

In vitro characterization of a novel series of platelet-derived growth factor receptor tyrosine kinase inhibitors.

In this report, we describe the discovery and characterization of a novel biarylhydrazone series of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors typified by the prototype WIN 41662 (3-phenyl-N1-[1-(4-pytidyl)pyrimidine]hydrazone). WIN 41662 inhibited PDGF-stimulated autophosphorylation of PDGF receptors from human vascular smooth muscle cells (hVSMC) with an IC50 value of 60 nM. The inhibitor appeared to be competitive with respect to substrate (Mn(2+)-ATP), having a calculated Ki of 15 +/- 5 nM. WIN 41662 was approximately 500-fold more potent in inhibiting the PDGF receptor tyrosine kinase than the p56lck tyrosine kinase. It was inactive against other serine/threonine and tyrosine kinases tested. WIN 41662 produced concentration-dependent inhibition of PDGF-stimulated receptor autophosphorylation in intact hVSMC with an IC50 < 100 nM. Intracellular Ca2+ mobilization and cell proliferation were events that occurred in hVSMC subsequent to PDGF receptor activation. WIN 41662 inhibited PDGF-stimulated Ca2+ mobilization and cell proliferation ([3H]TdR incorporation) with IC50 values of 430 nM and 2.3 microM, respectively. These effects appeared to be specifically related to PDGF receptor tyrosine kinase inhibition since WIN 41662 was not cytotoxic (in vitro) and since WIN 72039, a close structural analog that does not inhibit PDGF receptor tyrosine kinase, also did not inhibit PDGF-stimulated receptor autophosphorylation, Ca2+ mobilization, or hVSMC proliferation. Thus, WIN 41662 is representative of a novel class of selective PDGF receptor tyrosine kinase inhibitors that inhibit PDGF-regulated secondary events in intact cells.

Zaprinast increases cyclic GMP levels in plasma and in aortic tissue of rats.

The purpose of this study was to determine if significant relationships exist between plasma and aortic cyclic GMP (cGMP) levels and pharmacodynamic effect after the i.v. administration of the cGMP-selective phosphodiesterase inhibitor zaprinast to conscious, spontaneously hypertensive rats. Zaprinast dose-dependently increased plasma and aortic cGMP levels at 10, 18 and 30 mg/kg and decreased mean arterial blood pressure (MAP) at 18 and 30 mg/kg. The concentrations of cGMP in the plasma and in the aorta were significantly correlated (r = 0.765, P < 0.0001). The changes in MAP were significantly correlated to aortic (r = -0.750, P < 0.0001) and plasma (r = -0.762, P < 0.0001) cGMP levels. We conclude that plasma cGMP may be an index of cGMP-selective phosphodiesterase inhibition in vivo.

Zaprinast, but not dipyridamole, reverses hemodynamic tolerance to nitroglycerin in vivo.

Hemodynamic tolerance to nitroglycerin was developed in spontaneously hypertensive rats following 2-3 days of pretreatment with 100 mg/kg of nitroglycerin administered s.c. 3 times/day. Tolerance was evaluated both in vivo, by administering ascending bolus doses of nitroglycerin of 1-300 micrograms/kg i.v., and ex vivo in isolated, denuded aortic vascular rings by exposure to ascending concentrations of nitroglycerin of 0.0003-100 microM. Tolerance was observed as a significant blunting of the hypotensive and vasorelaxant effect of nitroglycerin. Co-incubation of tolerant aortic rings and pretreatment of tolerant SHR with 10 microM and 0.1-10 mg/kg zaprinast, respectively, resulted in full restoration of the vasorelaxant and hypotensive effect of nitroglycerin. Zaprinast partially reversed hemodynamic tolerance at 0.01 mg/kg. Conversely, dipyridamole (10 microM) reversed tolerance ex vivo, but was ineffective in reversing tolerance in vivo at pretreatment doses of 30 and 60 mg/kg. Following a 100-micrograms/kg i.v. challenge dose of nitroglycerin, aortic cyclic guanosine monophosphate (cGMP) levels were lower in nitroglycerin tolerant SHR when compared to non-tolerant SHR. Pretreatment of tolerant SHR with 10 mg/kg zaprinast restored the increase in cGMP levels to nitroglycerin to that seen in non-tolerant SHR. Conversely, dipyridamole (30 mg/kg) pretreatment was not effective in restoring cGMP levels. These data therefore suggest that reversal of hemodynamic tolerance in vivo is related to restoration of changes in vascular cGMP levels. Zaprinast, a selective cGMP phosphodiesterase inhibitor, effectively reverses tolerance and dipyridamole, a rather non-selective inhibitor, does not.

Cardiovascular activity of WIN 65579, a novel inhibitor of cyclic GMP phosphodiesterase 5.

This study describes the phosphodiesterase inhibitory potency and cardiovascular actions of WIN 65579 (1-cyclopentyl-3-ethyl-6-(3-ethoxy-4-pyrridyl)-1H-pyrazolo[3,4-d]p yrimidin-4-one), a potent, new cGMP phosphodiesterase 5 inhibitor. WIN 65579 is a competitive inhibitor of phosphodiesterase 5, with IC50 values of 2-3 nM for phosphodiesterase 5 from human or canine vascular sources. WIN 65579 has low affinity for phosphodiesterases 1, 2 and 3 (IC50 > 3-10 microM), and is somewhat selective for phosphodiesterase 4 (IC50 approximately 100 nM). WIN 65579 is an endothelial-dependent relaxant of rat aortic smooth muscle (EC50 = 60 nM) and lowers mean arterial blood pressure in conscious spontaneous hypertensive rats following intravenous or oral dosing. WIN 65579 also increases plasma cGMP levels, and reinstates vascular responsiveness to nitroglycerin in conscious rats that are nitroglycerin-tolerant. These data show that WIN 65579 is one of the more potent phosphodiesterase 5 inhibitors, and that WIN 65579 possesses cardiovascular activities consistent with vascular phosphodiesterase 5 inhibition in vivo.

Ocular effects of topical administration of the phosphodiesterase III inhibitor milrinone in rabbits and cats.

Increases in intracellular cAMP levels have previously been shown to decrease intraocular pressure (IOP) and increase ocular blood flow (OBF). However, the ocular effects of milrinone, which increases intracellular cAMP levels via selective cAMP PDE III inhibition, have not been investigated. The purpose of this study was to investigate the ocular effects of topically administered milrinone at different concentrations in rabbits and cats. When compared to vehicle in conscious rabbits, topical administration of milrinone at 0.03% decreased IOP (-14.1 +/- 2.6% vs. -7.4 +/- 3.7%, max. changes expressed as mean +/- SEM), at 0.1% increased IOP (10.4 +/- 8.5% vs. -1.7 +/- 4.1%), and at 0.01% and 1% did not significantly affect IOP. Neither pupil size nor central corneal thickness were affected by milrinone. Additionally, there were no signs of inflammation and no effects on corneal clarity. In anesthetized cats, topical administration of milrinone at 0.01-0.3% increased OBF (38.9 +/- 6.0% for milrinone vs. -7.4 +/- 4.4% for vehicle), and at 0.03%-0.3% decreased mean arterial pressure (-19.0 +/- 5.6 vs. 3.0 +/- 4.1 mmHg) in a dose-related manner. The durations of OBF enhancement (1.5-2.5 h) and MAP reduction (less than 30 min to 2 h) were also dose-dependent. In conclusion, milrinone induced biphasic IOP effects: IOP was decreased at 0.03% but increased at 0.1%. Milrinone at 0.01% increased OBF, possibly via a local vasodilator effect, and at 0.03-0.3% increased OBF, possibly via local and systemic effects. These data suggest that cAMP PDE III inhibitors such as milrinone may have efficacy as agents which enhance ocular blood flow following topical ocular application.

Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes.

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7x10(7) cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.

Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells.

The effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane were examined with cultured S49 lymphoma cells. The beta-adrenergic receptor-coupled adenylate cyclase system was used as a probe of the functional properties of the plasma membrane. Steady-state fluorescence anisotropy of diphenylhexatriene and the lipid composition of the plasma membrane were used as probes of the physical properties of the membrane. Cells were grown under conditions such that the concentration of ethanol in the growth medium remained stable and oxidation of ethanol to acetaldehyde was not detected. Chronic exposure of S49 cells to 50 mM ethanol or growth of cells at elevated temperature resulted in a decrease in adenylate cyclase activity. There were no changes in the density of receptors or in the affinity of beta-adrenergic receptors for agonists or antagonists following chronic exposure to ethanol. The fluorescence anisotropy of diphenylhexatriene was lower in plasma membranes prepared from cells that had been treated with 50 mM ethanol than in membranes prepared from control cells. However, this change was not associated with changes in the fatty acid composition or the cholesterol to phospholipid ratio of the plasma membrane. There was a small but statistically significant decrease in the amount of phosphatidylserine and an increase in the amount of phosphatidylethanolamine. These changes cannot account for the decrease in anisotropy. In contrast to the effect of ethanol, a decrease in adenylate cyclase activity following growth of S49 cells at 40 degrees C was not associated with a change in anisotropy.

Post-receptor modulation of the effects of cyclic AMP in isolated cardiac myocytes.

The actions of cyclic AMP are subject to several levels of post-receptor modulation in cardiac tissue. Isoproterenol and prostaglandin E1 both stimulate cAMP accumulation, but only isoproterenol causes activation of particulate cAMP-dependent protein kinase, leading to activation of phosphorylase kinase and glycogen phosphorylase, and inhibition of glycogen synthase. Through the use of isolated, adult ventricular myocytes, we have determined that the hormone-specific activation of glycogen phosphorylase is due to subcellular compartmentation of cAMP. There is some evidence that cyclic nucleotide phosphodiesterases, whose activity is stimulated by alpha 1-adrenergic agonists in isolated myocytes, may have a role in compartmentation. Phosphoinositide hydrolysis is stimulated by alpha 1 and muscarinic agonists, presumably leading to activation of protein kinase C, which in turn has multiple effects on hormone-sensitive adenylate cyclase.

Effects of ethanol in vitro on the beta adrenergic receptor-coupled adenylate cyclase system.

The effects of ethanol on the beta adrenergic receptor-coupled adenylate cyclase system were examined in vitro using membranes prepared from S49 lymphoma cells. Ethanol caused a dose-dependent increase in adenylate cyclase activity in membranes prepared from wild-type cells when the activity was measured in the presence of GTP. Activity measured in the presence of isoproterenol was also increased by ethanol, but the fold-stimulation by isoproterenol was lower in the presence of ethanol. Ethanol also shifted the dose-response curve for stimulation of the enzyme by isoproterenol to the right. This shift was due to a decrease in the affinity of the beta adrenergic receptor for isoproterenol. A decrease in the affinity of the receptor for the antagonists [125I]iodopindolol and propranolol was also observed, but the magnitude of this effect was less than that seen with the agonist isoproterenol. The density of binding sites for [125I]iodopindolol was not affected by ethanol. Dose-response curves for NaF and guanosine-5'-O-(3-thiotriphosphate), both of which stimulate adenylate cyclase activity through an effect on the stimulatory guanine nucleotide-binding protein (Gs), were shifted to the left by the addition of ethanol. In membranes prepared from the CYC- variant of S49 cells, which lacks the alpha subunit of Gs, guanosine-5'-O-(3-thiotriphosphate) inhibited forskolin-stimulated adenylate cyclase activity. The inhibition by guanosine-5'-O-(3-thiotriphosphate) was not affected by ethanol. In membranes prepared from both wild-type and CYC- S49 cells, ethanol inhibited forskolin-stimulated adenylate cyclase activity. Whereas the inhibition of this activity by GTP was greatly attenuated in membranes prepared from CYC- S49 cells which had been pretreated with pertussis toxin, the inhibition by ethanol was not affected by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct profiles of phosphodiesterase isozymes in cultured cells derived from nonpigmented and pigmented ocular ciliary epithelium.

Alterations in either cyclic AMP (cAMP) or cyclic GMP (cGMP) may modulate the production of aqueous humor by the ciliary epithelium of the eye, thereby affecting intraocular pressure. We have found distinct profiles of phosphodiesterase (PDE) isozyme activity in cultured cells derived from bovine pigmented ciliary epithelium (PE) and cells derived from human nonpigmented ciliary epithelium (NPE), as well as corresponding differences in the effects of selective PDE inhibitors on the accumulation of cAMP and cGMP. In NPE cells, but not in PE cells, the major peak of PDE activity was stimulated by Ca++/calmodulin-stimulated (PDE I), and hydrolyzed both cAMP and cGMP. In contrast, PE cells contained a cGMP-specific PDE V not found in NPE cells. Rolipram, a selective inhibitor of PDE IV, was more potent and effective than the selective PDE III inhibitor CI-930 at potentiating intracellular cAMP accumulation in both cell types. Zaprinast, a selective inhibitor of PDE V, potentiated cGMP accumulation in PE but not in NPE cells. The results suggest that selective PDE inhibitors may modulate aqueous humor production by pigmented and nonpigmented ciliary epithelium, the two cell types may have different functional roles, and selective modulation of their functions may be possible. Furthermore, there may be distinct roles for intracellular calcium in regulating cGMP and cAMP in pigmented vs. nonpigmented ciliary epithelial cells.

Cellular distribution of phosphodiesterase isoforms in rat cardiac tissue.

We have resolved multiple forms of cyclic nucleotide phosphodiesterase (PDE) in whole rat ventricle and in isolated rat ventricular myocytes by use of anion-exchange high-performance liquid chromatography. One major form, the soluble calmodulin-stimulated PDE, is apparently absent from isolated myocytes. We discern four peaks of PDE activity (designated A-D in the order of their elution) in a soluble fraction obtained from whole rat ventricle. Peak A is stimulated twofold to threefold by the addition of calcium and calmodulin (Ca2+/CalM) and preferentially hydrolyzes cGMP over cAMP (in the presence of Ca2+/CalM, KmcGMP = 1.5 microM, KmcAMP = 17 microM). Peak B has similar affinities for both cAMP and cGMP (half-maximum velocities achieved at 30 microM substrate) and demonstrates positive cooperativity with cAMP but not with cGMP. The hydrolysis of cAMP by peak B is stimulated by cGMP at substrate concentrations up to 20 microM; the maximum effect is seen at 1 microM cAMP (25-fold stimulation by 3 microM cGMP). This pattern of stimulation by cGMP results from two kinetic changes: an increase in the enzyme's apparent affinity for cAMP (apparent KmcAMP decreases from 33 to 11 microM) and the abolition of positive cooperativity. Peaks C and D selectively hydrolyze cAMP, are not stimulated by Ca2+/CalM or cGMP, and differ in their affinities for substrate (peak C, apparent KmcAMP = 7.2 microM; peak D, 0.44 microM). In addition, peak D is much more sensitive than peak C to inhibition by cGMP, cilostamide, rolipram, and milrinone. Ro20-1724 is a slightly more potent inhibitor of peak D than of peak C. Peak D appears to consist of two different enzyme activities, one inhibited by cGMP, cilostamide, and cardiotonic drugs and the other potently inhibited by rolipram. In contrast to whole ventricle, the soluble fraction of isolated rat ventricular myocytes lacks peak A. Three major peaks in myocytes are entirely analogous to peaks B, C, and D of whole ventricle in terms of the NaCl concentration at which they elute, substrate affinities, and stimulation or inhibition by various drugs and effectors. We conclude that the soluble Ca2+/CalM-stimulated PDE in whole rat ventricle is present in nonmyocyte cells.

Adenosine 3',5'-cyclic monophosphate attenuates neutrophil-mediated increase in endothelial permeability.

We studied the effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) and cholera toxin (CT) on 125I-labeled albumin flux across confluent monolayers of bovine pulmonary microvessel endothelial cells (BMVEC) grown on polycarbonate filters (10(5) BMVEC/filter). 8-BrcAMP and CT increased endothelial adenosine 3',5'-cyclic monophosphate (cAMP) concentrations about twofold. Polymorphonuclear leukocytes (PMN) were layered on BMVEC monolayers (ratio of 10:1) and activated with phorbol 12-myristate 13 acetate (PMA; 5 x 10(-9) M). Transendothelial 125I-labeled albumin clearance rate was measured to determine the endothelial permeability alterations. Activation of PMN in control monolayers resulted in an increase in transendothelial 125I-labeled albumin clearance rate from 0.090 +/- 0.011 to 0.37 +/- 0.06 microliters/min (P < 0.01). Treatment of endothelial monolayers with 8-BrcAMP (10(-3) M) significantly attenuated the increase in endothelial permeability after PMN activation (transendothelial 125I-labeled albumin clearance rate increased to 0.19 +/- 0.03 microliters/min; P < 0.01). Pretreatment of BMVEC monolayers with CT (10(-8) M) for 3 h before PMN activation prevented the PMN-mediated increase in endothelial permeability (125I-labeled albumin clearance rate only increased to 0.13 +/- 0.018 microliters/min). To simulate the effect of PMN activation, hydrogen peroxide (H2O2) was added directly onto BMVEC; both 8-BrcAMP and CT were shown to reduce the H2O2-mediated increase in endothelial permeability. 8-BrcAMP and CT pretreatment did not prevent PMN adhesion to BMVEC monolayer and superoxide anion and H2O2 production after PMA activation of PMN. We conclude that increased endothelial cAMP concentration prevents PMN-mediated endothelial injury by an action of the cyclic nucleotide on endothelial cells.

Enrichment and analysis of desmosine and isodesmosine in biological fluids.

A method has been developed for the enrichment and analysis of the elastin crosslinks, desmosine and isodesmosine, in biological fluids and tissues. It is adapted from published methods, offering improved recovery, sensitivity, resolution, and speed of analysis. Samples were hydrolyzed in 6 M HCl, after which the desmosines were enriched by CF1 cellulose chromatography and analyzed by HPLC with a C18 column. Isodesmosine and desmosine were quantitated based on absorbance at 275 nm, with a limit of detection of approximately 30 pmol and recovery of approximately 66% in urine. Their tR values on our HPLC system were approximately 9 and 12 min, respectively. This method was used to evaluate the daily and weekly variation in the concentrations of desmosine and isodesmosine in human urine. The results suggest that this method can be used to process large numbers of biological samples for analysis of desmosine and isodesmosine.

Resolution of soluble rat cardiac phosphodiesterases by high performance liquid chromatography.

A high performance liquid chromatographic method has been developed to separate isozymes of cyclic nucleotide phosphodiesterase. The method employs a polymer-based anion exchange column eluted with a sodium chloride gradient. Compared to traditional chromatography over DEAE-cellulose, the method is more rapid (30 min), dilutes the sample less, achieves better resolution of kinetically distinct forms, may be applied to as little as 200 micrograms of tissue protein and is appropriate for analytical use.

Differential effects of cytosolic modulators of fluoride-stimulated adenylate cyclase activity in striatum and cerebral cortex.

The effects of sodium fluoride (NaF) and ethanol on adenylate cyclase activity were investigated in mouse and rat striatum and cerebral cortex. In a crude homogenate of striatum, NaF (10 mM) had no effect on adenylate cyclase activity even though guanyl-5'-yl-imidodiphosphate (GppNHp) increased enzyme activity by two-fold. Addition of 300 mM ethanol increased basal and GppNHp-stimulated activity and allowed expression of an effect of NaF. Stimulation of adenylate cyclase activity by NaF was also observed after sedimentation and resuspension of membranes. Readdition of the supernatant to washed membranes caused a decrease in maximal NaF-stimulated activity without any change in the concentration of NaF required for half-maximal stimulation. The inhibitory effect of the supernatant was resistant to heat but was eliminated by the addition of ethanol or perchloric acid. Inhibition of NaF-stimulated adenylate cyclase activity in striatal tissue was also observed when assays were carried out in the presence of a 20,000 X g supernatant prepared from cerebral cortex. NaF-stimulated activity in cortical tissue was, on the other hand, enhanced in the presence of a 20,000 X g supernatant prepared from either cortex or striatum. This suggests that there is a basic difference between the adenylate cyclase systems of these tissues.

Comparison of urinary desmosine excretion in patients with chronic obstructive pulmonary disease or cystic fibrosis.

Neutrophil elastase is involved in the pathogenesis of several pulmonary diseases; a strategy for monitoring in vivo elastase activity is to measure changes in biochemical markers. The objective of this study was to determine whether differences in the urinary excretion of the elastin crosslinks, desmosine and isodesmosine (which are unique amino acid products of elastase activity), could be discerned between groups of patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF), and non-diseased, age-matched controls. Twenty-four-hour urine collections were analysed to eliminate variations in excretion throughout the day, and urine was collected on four separate days in 29-31 subjects/group to investigate the variability in desmosines excretion among the groups. Both sets of patient populations had significantly more variable desmosines readings (higher standard deviations) relative to their respective age-matched control group. The means for three adult groups (COPD, controls and a COPD-smoker subset) ranged from 28.4 to 35.5 pmol desmosines/mg creatinine and there were no differences among the groups. Values in children were higher: 55 pmol desmosines/mg creatinine in the non-CF children and 77 pmol desmosines/mg creatinine for the CF group (P<0.01 vs. age-matched controls). The results of this study show that urinary desmosines, as a surrogate marker for enhanced elastase activity, are more highly variant in both patient populations relative to age-matched controls, and an overall increase in the mean value is further observed in patients with cystic fibrosis.

Species-dependent pharmacodynamic effects of the selective low Km cyclic AMP phosphodiesterase III inhibitors WIN 58993 and WIN 62005.

We describe the biochemical and pharmacologic effects of two novel fused pyridinones derived from milrinone: WIN 58993 and WIN 62005. Both WIN 58993 and WIN 62005 competitively inhibit cyclic GMP-inhibitable low Km cyclic AMP phosphodiesterase (PDE III) from rat heart and canine aorta with Ki values of 25 +/- 3 and 26 +/- 5 nM, respectively, and are selective (at least 160-fold) for PDE III inhibition relative to other PDE isozymes. WIN 58993 and WIN 62005 were given to conscious, chronically instrumented rats and dogs intravenously (i.v.) or perorally (p.o.). Because the doses of WIN 58993 and WIN 62005 required to decrease mean arterial blood pressure (MAP) by 20% were estimated to be 0.9 and 0.7 mg/kg, respectively, the compounds appear to be equipotent after acute i.v. administration in rats. However, the duration of the depressor responses in rats apparently differs since MAP remained significantly decreased 6 h after i.v. or p.o. administration of WIN 58993, but returned to control levels < or = 4 h after administration of WIN 62005. WIN 58993 may be slightly less potent than WIN 62005 after acute i.v. administration to dogs since significant increases in left ventricular (LV)dP/dtmax first occurred at doses of 0.1 and 0.03 mg/kg, respectively. LVdP/dtmax significantly increased in 30 min and returned to baseline 3 h after p.o. administration of 1 mg/kg WIN 58993. After p.o. administration of 1 mg/kg WIN 62005. LVdP/dtmax was significantly increased in 30 min and remained increased for at least 6 h. These data suggest that WIN 58993 and WIN 62005 are potent, selective, p.o.-active inhibitors of PDE III.(ABSTRACT TRUNCATED AT 250 WORDS)