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.

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.

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.

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.

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.

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)

Pharmacologic and pharmacodynamic effects of the selective low Km cyclic AMP phosphodiesterase III inhibitors WIN 63291 and WIN 62582.

We describe the biochemical, pharmacologic, and in vivo pharmacodynamic profiles of two novel inhibitors of the cyclic GMP-inhibitable, low Km cyclic AMP phosphodiesterase (PDE) III; WIN 63291, a 6-quinolinyl analogue of the prototypic PDE III inhibitor milrinone and WIN 62582, an imidazopyridinone. Both WIN 62582 and WIN 63291 competitively inhibit PDE III from rat, dog, and human heart and from rat and canine aorta with IC50 values of 5-37 and 55-80 nM, respectively; the IC50 values for milrinone ranged from 300 to 520 nM. WIN 62582 and WIN 63291 are at least 1,000-fold selective for PDE III relative to inhibition of PDE isozymes I, II, IV, and V. We evaluated WIN 62582 and WIN 63291 in conscious rats and dogs after intravenous (i.v.) and oral (p.o.) administration. The dose of WIN 62582 required to reduce mean arterial blood pressure (MAP) by 20% (ED20) in rats was 1.8 mg/kg, with a pharmacodynamic duration of action of approximately 2 h. In comparison, the estimated i.v. ED20 for WIN 63291 in rats was 0.4 mg/kg, with a pharmacodynamic duration of action > 6 h. In conscious dogs, the i.v. doses of WIN 62582 and 63291 required to increase left ventricular (LV)dP/dtmax significantly were 0.1 and 0.01 mg/kg, respectively. In dogs, WIN 63291 0.1 mg/kg p.o. increased LVdP/dtmax by 86% in 30 min; LVdP/dtmax remained increased by 60% for at least 6 h. In comparison, WIN 62582, 0.3 mg/kg p.o., increased LVdP/dt by 56% in 30 min and remained increased by 40% at 6 h.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Reversal of nitroglycerin tolerance in vitro by the cGMP-phosphodiesterase inhibitor zaprinast.

Following in vitro exposure of rat aortic rings to 550 microM nitroglycerin for 1 h, tolerance was demonstrated by a significant increase in EC50 values for nitroglycerin-induced relaxation. However, cross-tolerance to sodium nitroprusside was not observed. Co-incubation of aortic rings with the cGMP-phosphodiesterase (cGMP-PDE) inhibitor zaprinast (10 microM), during incubation with 550 microM nitroglycerin, did not prevent the development of tolerance. However, the addition of 0.30 or 10 microM zaprinast to tolerant aortic rings did restore responsiveness to nitroglycerin. The increase in cGMP in tolerant aortic rings in response to 300 nM nitroglycerin (2-4 fmol/micrograms) was significantly less than that observed for non-tolerant rings (6.6-12 fmol/micrograms), but cGMP levels were restored in tolerant rings by zaprinast (7-12 fmol/micrograms). These data suggest that inhibition of vascular cGMP-PDE activity does not prevent the development of tolerance in vitro, but does reverse the loss of vasorelaxant potency to nitroglycerin via restoration of intracellular cGMP levels.

Identification of a specific radioligand for the cardiac rapidly activating delayed rectifier K+ channel.

Class III antiarrhythmic drugs show promise as effective treatments for the suppression of potentially lethal cardiac arrhythmias. Dofetilide (UK-68,798), is a potent class III antiarrhythmic agent that is presently under clinical investigation. The objective of this study was to determine whether [3H]dofetilide could be used as a specific radioligand for the rapidly activating delayed rectifier K+ channel of the heart. We find that [3H]dofetilide binds to high-affinity sites on guinea pig cardiac myocytes. Competition studies using unlabeled dofetilide indicate that binding is characterized by an IC50 of 100 +/- 30 nM (mean +/- SD, n = 13). Scatchard analyses of binding indicate a Kd of 70 +/- 6 nM and a maximal binding capacity of 0.30 +/- 0.02 pmol/mg protein. [3H]Dofetilide is displaced from guinea pig myocytes by dofetilide, clofilium, quinidine, sotalol, and sematilide with a rank order of potency that correlates with functional blockade of the rapidly activating delayed rectifier K+ current (correlation coefficient, 0.951; slope, 0.99 +/- 0.19; p = 0.014). High-affinity [3H]dofetilide binding is not detected in rat myocytes, which are devoid of delayed rectifier K+ current. We conclude that [3H]dofetilide specifically binds to sites associated with the rapidly activating delayed rectifier K+ channel of guinea pig myocardium.

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.

Troponin T isoform expression in the normal and failing human left ventricle: a correlation with myofibrillar ATPase activity.

The expression of troponin T, a thin filament regulatory protein, was examined in normal and failing left ventricles. The samples were obtained from the hearts of patients with severe heart failure who were undergoing cardiac transplantation, and from normal adult hearts that could not be used for transplantation. Western blots of the myofibrillar proteins demonstrated two isoforms, troponin T 1 (TnT1) and troponin T 2 (TnT2). TnT2 is expressed at significantly higher levels in failing hearts (p less than 0.004). Western blots of two-dimension SDS-PAGE gels resolved two dominant spots of TnT1 and of TnT2 and several minor troponin T species. Alkaline phosphatase treatment markedly decreased the sizes of the two acidic spots while increasing the two more basic spots by a comparable amount. Myofibrillar ATPase activity had an inverse and negative linear relationship (r = 0.7, p less than 0.02) with the myofibrillar percentage of total troponin T comprised of TnT2. In that heart failure in these transplant patients had multiple bases, we propose that rather than a cause of heart failure, the disease-associated changes in troponin T isoform expression are an adaptation to abnormal myocardial function.

Cardiovascular cyclic nucleotide phosphodiesterases and their role in regulating cardiovascular function.

We have described five phosphodiesterase (PDE) isozymes that can be found in cardiac and vascular smooth muscle of animals and humans. Much of the evidence for the role that these isozymes have in the regulation of cellular processes has been generated through, or awaits, the identification of selective and potent PDE inhibitors. While selective inhibitors of the cGMP-inhibitable (cGi)-PDE isozyme have been approved for use in the acute treatment of heart failure, selective inhibitors of the cGMP-PDE have not been extensively explored as potential candidates for the treatment of cardiovascular diseases. More potent selective inhibitors of the cGMP-PDE isozyme are needed to determine whether these pharmacological potentiators of EDRF and ANP will be useful in the therapy of angina, hypertension or heart failure.

Troponin T isoform expression in humans. A comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle.

The expression of troponin (Tn) T, a thin-filament regulatory protein, was examined in left ventricular myocardium from normal and from failing adult human hearts. The differences in isoform expression between normal and failing myocardium led us to examine the ontogenic expression of TnT in human striated muscle. Left ventricular samples were obtained from patients with severe heart failure undergoing cardiac transplantation and normal adult organ donors. Fetal muscle was obtained from aborted fetuses after 14-15 weeks of gestation, and adult skeletal muscle was obtained from surgical biopsies. Western blots of normal and failing adult heart proteins demonstrated that two isoforms, TnT1 and TnT2, are expressed in different amounts, with TnT2 being significantly greater in failing hearts (p less than 0.004). Western blots of two-dimensional gels of these proteins resolved two predominant spots of both TnT1 and TnT2 and several minor TnT species. Alkaline phosphatase treatment converted the two major spots of each isoform into the single more basic spots. A comparison of the ATPase activities and the TnT2 percentage of total TnT in individual failing and normal adult hearts demonstrated an inverse and negative relation (r = 0.7, p less than 0.02). In the fetal heart, four TnT isoforms were found, two of which had the same electrophoretic mobilities as the adult cardiac isoforms TnT1 and TnT2. Fetal skeletal muscle expressed two of the four fetal cardiac TnT isoforms, one of which comigrated with adult cardiac TnT1. These cardiac isoforms were expressed in low abundance in fetal skeletal muscle relative to seven fast skeletal muscle TnT isoforms. No cardiac isoforms were present in adult skeletal muscle. Because many etiologies caused heart failure in the transplant patients, we propose that the disease-associated increased expression of the TnT isoform TnT2 is an adaptation to the heart failure state and a partial recapitulation of the fetal expression of cardiac TnT isoforms.

N omega-nitro-L-arginine attenuates the accumulation of aortic cyclic GMP and the hypotension produced by zaprinast.

To determine if N omega-nitro-L-arginine (NNA), an inhibitor of the synthesis and/or release of endothelium-derived relaxing factor (EDRF), alters the response to zaprinast, a selective inhibitor of cyclic GMP (cGMP) phosphodiesterase, zaprinast (3-30 mg/kg) or vehicle (1 ml/kg) was given to conscious, spontaneously hypertensive rats (SHR) in a cumulative i.v. dose-response manner 30 min after pretreatment with NNA (1 or 3 mg/kg) or saline (1 ml/kg). Mean arterial pressure (MAP) was measured 5 min after each dose of zaprinast. Five minutes after the last dose of zaprinast (30 mg/kg), the rats were anesthetized with pentobarbital (25 mg i.v.). A segment of the abdominal aorta was freeze-clamped in situ and removed for the determination of cGMP levels. NNA (3 mg/kg) decreased basal aortic cGMP levels by 54% and increased MAP by 37 +/- 2 mm Hg. Zaprinast (30 mg/kg) increased aortic cGMP by 187% and decreased MAP by 49 +/- 4 mm Hg. NNA (3 mg/kg) reduced the accumulation of cGMP in aortic tissue (from 4.1 +/- 0.4 to 1.3 +/- 0.1 fmol/microgram protein) and attenuated the depressor response (from -49 +/- 4 to -31 +/- 4 mm Hg) produced by zaprinast. These data are consistent with the hypothesis that NNA inhibits the tonic release of EDRF and that the depressor effects of zaprinast are due, at least in part, to the potentiation of the vasodilator effects of EDRF in vivo. Moreover, since the changes in MAP produced by NNA and zaprinast were significantly correlated with cGMP levels in aortic tissue, the concentration of cGMP in vascular tissue may be a determinant of blood pressure in SHR.

Inhibition of low Km cGMP phosphodiesterases and Ca+(+)-regulated protein kinases and relationship to vasorelaxation by cicletanine.

In the present studies we sought to determine if cicletanine, which is an antihypertensive agent of unknown mechanism, could alter cGMP metabolism via inhibition of cGMP phosphodiesterases (PDE) in vascular smooth muscle. Cicletanine was determined to be a mixed (competitive, noncompetitive) inhibitor of both calmodulin-regulated and cGMP-specific PDEs from monkey aortic smooth muscle with Ki values of 450 to 700 microM. Cicletanine also potentiated vasorelaxation by the guanylate cyclase activators sodium nitroprusside and atrial natriuretic peptide in isolated rat aortas. Potentiation was not dependent upon the contractile agonists nor was it indomethacin-sensitive. Neither potentiation nor inhibition of cGMP PDEs was stereoselective. Methylene blue attenuated a component of cicletanine-induced vasorelaxation, but did not completely obviate relaxation. Both cicletanine and the cGMP-PDE inhibitor zaprinast potentiated sodium nitroprusside-mediated cGMP formation and relaxation, although the increase in cGMP content was markedly greater with zaprinast compared to cicletanine. In further studies, cicletanine did not potentiate cGMP activation of cGMP-dependent protein kinase, but did inhibit calmodulin-activated myosin light chain kinase and protein kinase C at relatively high concentrations (approximately 1 mM). In summary, these data demonstrate that cicletanine inhibits vascular cGMP PDEs, potentiates vasorelaxation, and to a limited extent, cGMP formation by guanylate cyclase activators in vascular smooth muscle. However, these relationships for cicletanine are dissimilar from the reference cGMP PDE inhibitor, zaprinast. Thus, other mechanisms may also contribute to the vasorelaxant action of cicletanine.