R.Allan Buchholz

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.

Inhibition of low Km cyclic GMP phosphodiesterases and potentiation of guanylate cyclase activators by cicletanine.

Cicletanine is an antihypertensive/vasorelaxant/natriuretic agent of unknown mechanism. We wished (a) to determine if cicletanine interacts with guanylate cyclase activators that modulate vasomotor tone and sodium balance [i.e., atriopeptin II (AP II), endothelium-derived relaxing factor (EDRF), and sodium nitroprusside (SNP)], and (b) to define the subcellular basis for this interaction by quantitating the effects of cicletanine on low Km cyclic GMP phosphodiesterase (PDE) activity. In phenylephrine-contracted rat aortic smooth muscle, the vasorelaxant potency of cicletanine was increased twofold in the presence of a threshold-relaxant concentration of AP II, and functional cyclic GMP PDE inhibition was also evident from the three- to sixfold potentiation by cicletanine of AP II- or SNP-induced vasorelaxation. Vasorelaxation produced by cicletanine was not endothelium dependent, however. In further studies, intravenous (i.v.) administration of cicletanine or the low Km cyclic GMP PDE inhibitor, zaprinast, decreased blood pressure (BP) ≤20% in conscious spontaneously hypertensive rats (SHR). These results are consistent with the additional finding that cicletanine inhibited Ca2+ -calmodulin (CaM) cyclic GMP PDE and zaprinast-sensitive cyclic GMP specific PDE over a concentration range (10–600 μM) similar to that for vasorelaxation. Thus, inhibition of low Km cyclic GMP PDEs by cicletanine may be partly responsible for the vasorelaxant effect of cicletanine as well as the potentiation by cicletanine of the vasorelaxant actions of guanylate cyclase activators. The extent to which this mechanism contributes to the antihypertensive efficacy of cicletanine has not yet been fully determined.

Reversal or nitroglycerin tolerance by the cGMP phosphodiesterase inhibitor zaprinast

In in vitro experiments, aortic rings (2-3mm) from Sprague-Dawley rats (250-350g) were incubated for 1 h with 550 μM nitroglycerin or vehicle (1.65% PEG-400), extensively rinsed, transferred to naive tissue baths, and subsequently tested for concentration-related vasorelaxation to nitroglycerin in the presence and absence of zaprinast. In in vivo experiments, spontaneously hypertensive rats (17 weeks) were injected with 100 mg nitroglycerin/kg body weight s.c., 3 times/day for 3 days. Decreases in mean arterial blood pressure (MAP) in response to nitroglycerin in the presence or absence of zaprinast were quantitated prior to the initiation of nitroglycerin injection (day 0), and after 3 days of nitroglycerin injection (fig. 1, bottom)

Sodium nitroprusside potentiates the depressor response to the phosphodiesterase inhibitor zaprinast in rats

To determine if the presence of an activator of guanylate cyclase alters the depressor response to a selective inhibitor of low Km cyclic GMP (cGMP) phosphodiesterase (PDE), zaprinast (3-30 mg/kg) was given i.v. to conscious, spontaneously hypertensive rats during a steady state of i.v. infusion of sodium nitroprusside (15 micrograms/kg per min). Sodium nitroprusside significantly increased the magnitude of the depressor response to zaprinast. In contrast, fenoldopam (20 micrograms/kg per min), an activator of adenylate cyclase, did not affect the depressor response to zaprinast. Zaprinast (10 mg/kg) significantly decreased mean arterial pressure (MAP) in rats given an infusion of sodium nitroprusside, an activator of soluble guanylate cyclase, at doses of 15 and 25 micrograms/kg per min but not at a dose of 5 micrograms/kg per min. However, in rats given atrial natriuretic peptide (ANP; 0.5, 1 and 2 micrograms/kg per min), an activator of particulate guanylate cyclase, zaprinast (10 mg/kg) did not affect MAP. In contrast to the potentiation of the depressor response to zaprinast, sodium nitroprusside (15 micrograms/kg per min) significantly attenuated the reductions in MAP produced by CI-930, a selective inhibitor of low Km cAMP PDE. It is concluded that sodium nitroprusside, but not ANP or fenoldopam, potentiates the depressor response to zaprinast. Furthermore, the potentiation of the depressor response to zaprinast is dependent upon the dose of sodium nitroprusside and is selective for zaprinast; the depressor response to CI-930 is attenuated by sodium nitroprusside.

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

Abstract 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 μg/kg i.v., and ex vivo in isolated, denuded aortic vascular rings by exposure to ascending concentrations of nitroglycerin of 0.0003–100 μM. 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 μM 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 μM) reversed tolerance ex vivo, but was ineffective in reversing tolerance in vivo at pretreatment doses of 30 and 60 mg/kg. Following a 100-μg/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.

Nω-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.

Lack of cross-tolerance between nitroglycerin and endothelium-derived relaxing factor-mediated vasoactive agents in spontaneously hypertensive rats

The purpose of this study was to determine whether cross-tolerance develops between nitroglycerin and endothelium-derived relaxing factor (EDRF)-mediated vasoactive agents in vivo. Spontaneously hypertensive rats (SHR) were made tolerant by pretreatment with high doses of nitroglycerin (100 mg/kg s.c., 3 times/day, for 3 consecutive days). The hypotensive effect of challenge doses of nitroglycerin (1, 10, 300, 100 micrograms/kg i.v.) was completely abolished in nitroglycerin-pretreated SHR. To evaluate cross-tolerance, the effects of the following EDRF-dependent vasoactive agents on blood pressure were determined in groups of nitroglycerin-pretreated and vehicle-pretreated SHR: acetylcholine, bradykinin and L-arginine. In addition, the hypotensive effects of zaprinast (M & B 22,928), a cyclic guanosine monophosphate (cGMP) phosphodiesterase inhibitor, and the hypertensive effects of the nitric oxide-synthase inhibitor N omega-nitro-L-arginine were also evaluated. In all cases, there was no difference in the effects of these agents on blood pressure when compared in nitroglycerin-pretreated (tolerant) and vehicle-pretreated (non-tolerant) SHR. The use of a variety of agents which modulate EDRF release or its effects by several different mechanisms suggests that cross-tolerance does not occur between nitroglycerin and EDRF in vivo.

Pharmacologic and Pharmacodynamic Effects of the Selective Low Km Cyclic AMP Phosphodiesterase III Inhibitors WIN 63291 and WIN 62582

Summary 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-qui-nolinyl analogue of the prototypic PDE III inhibitor milrinone and WIN 62582, an imidazopyridinone. Both WIN 62582 and WIN 63291 competitively inhibit PDE HI 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 ±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. These data suggest that WIN 62582 and WIN 63291 are potent, selective, orally active inhibitors of PDE III and have a relatively long duration of action in vivo. Although WIN 62582 is 10-fold more potent than WIN 63291 as an inhibitor of PDE III in vitro, WIN 63291 is a slightly more potent cardiovascular agent in vivo after i.v. and p.o. administration to conscious rats and dogs.

Hemodynamic Response of Conscious Rats and Dogs to the Protein Kinase C Inhibitor Staurosporine

Agonist-induced contraction of vascular smooth muscle appears to be mediated by Cat2+ influx through voltage dependent and independent channels and by receptor-linked, G-protein coupled activation of phospholipase C (1). Two intracellular messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG), are generated by phospholipase C catalyzed hydrolysis of inositol bisphosphate (2). Considerable biochemical and physiological evidence suggests that IP3-mediated mobilization of intracellular Ca2+ results in calcium-regulated phosphorylation of the 20,000 dalton myosin light chain and the initiation of smooth muscle contraction (3–5). However, the mechanism(s) responsible for sustaining isometric force in vascular smooth muscle are not fully understood. Several mechanisms have been proposed for the maintenance of smooth muscle tension, including the “latch bridge” state (6) and DAG activation of protein kinase C (PKC) (7). Evidence supporting the role of PKC in the maintenance of smooth muscle tone is further reviewed in an earlier chapter (8). Staurosporine has been identified as a very potent PKC inhibitor (9). The cardiovascular actions of staurosporine in vivo have not been fully characterized. Therefore, we examined the hemodynamic response to staurosporine in conscious, spontaneously hypertensive rats (SHR) and conscious normotensive dogs.

Calcium-Regulated Protein Kinases Low Km cGMP Phosphodiesterases: Targets for Novel Antihypertensive Therapy

The recent gain in knowledge over the last ten years on the intracellular mechanisms which regulate vascular smooth muscle tone has expanded opportunities for the potential discovery of novel vasodilator/antihypertensive agents. This review focuses on three intracellular enzyme systems: myosin light chain kinase (MLCK) and protein kinase C (PKC), which are Ca2+-regulated protein kinases implicated in the control of smooth muscle tone, and the cGMP phosphodiesterases (PDEs), which regulate the levels of cGMP in smooth muscle (Fig. 1).