Ursula Schindler

Release of nitric oxide from endothelial cells stimulated by YC‐1, an activator of soluble guanylyl cyclase

In this study we examined the endothelium‐dependent effect of YC‐1–a benzyl indazole derivative which directly activates soluble guanylyl cyclase (sGC)–on vascular relaxation and nitric oxide (NO) and guanosine‐3′,5′‐cyclic monophosphate (cyclic GMP) in endothelial cells. In preconstricted rat aortic rings with intact endothelium, YC‐1 produced a concentration‐dependent relaxation. However, the concentration response curve was shifted rightward to higher concentrations of YC‐1, when (i) the aortas were pre‐treated with L‐NG‐nitroarginine methylester (L‐NAME) or (ii) the endothelium was removed. Incubation of bovine aortic endothelial cells (BAEC) with YC‐1 produced a concentration‐dependent NO synthesis and release as assessed using a porphyrinic microsensor. Pre‐incubating cells with L‐NAME or with 8‐bromo‐cyclic GMP decreased this effect indicating that the YC‐1 stimulation of NO synthesis is due to an activation of nitric oxide synthase, but not to an elevation of cyclic GMP. No direct effect of YC‐1 on recombinant endothelial constitutive NO synthase activity was observed. The YC‐1 stimulated NO release was reduced by 90%, when extracellular free calcium was diminished. In human umbilical vein endothelial cells (HUVEC), YC‐1 stimulated intracellular cyclic GMP production in a concentration‐ and time‐dependent manner. Stimulation of cyclic GMP was greater with a maximum concentration of YC‐1 compared to calcium ionophore A23187. Similar effects were observed in BAEC and rat microvascular coronary endothelial cells (RMCEC). When HUVEC and RMCEC were pre‐treated with L‐NG‐nitroarginine (L‐NOARG), the maximum YC‐1 stimulated cyclic GMP increase was reduced by 50%. These results indicate, that beside being a direct activator of sGC, YC‐1 stimulates a NO‐synthesis and release in endothelial cells which is independent of elevation of cyclic GMP but strictly dependent on extracellular calcium. The underlying mechanism needs to be determined further.

Blood pressure-independent effect of long-term treatment with the soluble heme-independent guanylyl cyclase activator HMR1766 on progression in a model of noninflammatory chronic renal damage.

Nitric oxide formation is impaired in chronic renal failure. The renoprotective effects of a nonhypotensive dose of HMR1766, a direct activator of the heme enzyme soluble guanylyl cyclase was studied in comparison to an ACE-i in the remnant kidney model. Male Sprague-Dawley rats were subtotally nephrectomized (SNX) or sham operated (sham) and left untreated or started on treatment with HMR1766 or ACE-i in non-hypotensive doses. BP, albumin excretion and parameters of renal damage were analyzed. After a 12-week study, urinary albumin excretion was significantly higher in untreated SNX than in sham; this increase was prevented by ACE-i and ameliorated by HMR1766. Relative kidney and left ventricular weight were significantly higher in untreated SNX compared to sham; these changes were completely prevented by HMR1766. In untreated SNX, glomerulosclerosis (1.02 ± 0.13) was significantly higher than in sham (0.12 ± 0.04), SNX+HMR1766 (0.27 ± 0.04) and SNX+ACE-i (0.46 ± 0.06). Tubulointerstitial changes went in parallel. Increased glomerular cell number after SNX (71.5 ± 14 vs. 60 ± 7.3 in sham) was prevented by HMR1766 (55.7 ± 7.3), but not by ACE-i (66.6 ± 9). The results document beneficial BP-independent HMR1766 effects on kidney structure and urinary albumin excretion in a noninflammatory model of renal failure and may argue for a novel therapeutic principle.

Inhibition of Na+–H+ exchange by cariporide reduces inflammation and heart failure in rabbits with myocardial infarction

The aim of this study was to assess the effects of the Na+–H+ exchange inhibitor cariporide on left ventricular (LV) morphology and function as well as inflammation in rabbits with heart failure. Rabbits with myocardial infarction (MI) and sham controls were randomized to receive either standard chow or chow supplemented with cariporide for 9 weeks. LV morphology was determined by echocardiography. LV systolic and diastolic function was assessed under load‐dependent and ‐independent conditions by analysis of LV pressure–volume loops using piezo‐electric crystals. Plasma concentrations of C‐reactive protein and aldosterone were measured. Rabbits with MI developed LV dilatation that was reduced by cariporide. Systolic and diastolic LV function was impaired in rabbits with MI when compared to sham, as indicated by a decreased dP/dtmax (MI: 3537±718 mmHg s−1, sham: 5839±247 mmHg s−1, P<0.05), the load‐independent preload recruitable stroke work (PRSW)(MI: 22±7 mmHg, sham: 81±23 mmHg, P<0.05) and a reduction in the time constant of relaxation tau (τ) (MI: 27±1 ms, sham: 17±1 ms, P<0.05), and significantly improved by cariporide (dP/dtmax: 4586±374 mmHg s−1, PRSW: 67±18 mmHg, τ: 20±2 ms; P<0.05 vs MI/control). Induction of MI was associated with an increase in aldosterone and CRP, indicating activation of the neurohormonal and the inflammatory system that were largely reduced by cariporide. Cariporide improves LV morphology and function post MI and suppresses inflammation and neurohormonal activation in congestive heart failure (CHF). Na+–H+ exchange inhibition may represent a new pharmaceutical approach for the treatment of CHF.

Role of activators of ferric sGC in cardiovascular disease

Soluble guanylyl cyclase (sGC), the key transducer of nitric oxide (NO) signalling in vascular smooth muscle cells and platelets, exists in two redox forms: the NO-sensitive ferrous heme-iron (Fe(II)) form and a NO-insensitive oxidized form containing the ferric heme-iron (Fe(III)). HMR1766 (proposed INN ataciguat sodium) reversibly stimulates the oxidized form of sGC (crude and purified) in various species and organs to about 10 to 30 % of the maximal activity achieved with NO donors. Acute treatment with HMR1766 has anti-ischemic (acute coronary stenosis) and anti-thrombotic (coronary thrombosis) effects in dog models at doses that did not influence arterial blood pressure or other hemodynamic parameters.

Effects of combined inhibition of the Na+–H+ exchanger and angiotensin‐converting enzyme in rats with congestive heart failure after myocardial infarction

We investigated the single vs the combined long‐term inhibition of Na+–H+ exchanger‐1 (NHE‐1) and ACE in rats with congestive heart failure induced by myocardial infarction (MI). Rats with MI were randomized to receive either placebo, cariporide (3000 p.p.m. via chow), ramipril (1 mg kg−1 day−1 via drinking water) or their combination for 18 weeks starting on day 3 after surgery. Cardiac morphology and function was assessed by echocardiography and by means of a 2.0 F conductance catheter to determine left ventricular (LV) pressure volume relationships. MI for 18 weeks resulted in an increase in LV end‐diastolic diameter (LVDed) in the placebo‐treated group when compared to sham (placebo: 1.1±0.04 cm; sham: 0.86±0.01; P<0.05). Combined inhibition of NHE‐1 and ACE, but not the monotherapies, significantly reduced LVDed (1.02±0.02 cm). Preload recruitable stroke work (PRSW), dp/dtmax (parameter of systolic function) and end‐diastolic pressure volume relationship (EDPVR, diastolic function) were significantly impaired in placebo‐treated MI group (PRSW: 39±7 mmHg; dp/dtmax: 5185±363 mmHg s−1; EDPVR: 0.042±0.001 mmHg μl−1; all P<0.05). Cariporide treatment significantly improved PRSW (64±7 mmHg), dp/dtmax (8077±525 mmHg s−1) and EDPVR (0.026±0.014 mmHg μl−1), and reduced cardiac hypertrophy in rats with MI. Combined inhibition of NHE‐1 and ACE had even a more pronounced effect on PRSW (72±5 mmHg) and EDPVR (0.026±0.014 mmHg μl−1), as well as cardiac hypertrophy that, however, did not reach statistical significance compared to cariporide treatment alone. The NHE‐1 inhibitor cariporide significantly improved LV remodeling and function in rats with congestive heart failure induced by MI. The effect of cariporide was comparable or tended to be stronger (e.g. systolic function) compared to ramipril. Combined treatment with cariporide and ramipril tended to be more effective on LV remodeling in rats with heart failure than the single treatments. Thus, inhibition of the NHE‐1 may be a promising novel therapeutic approach for the treatment of congestive heart failure.

Cariporide, a selective Na+/H+ exchange inhibitor, decreases liver fibrosis in the bile duct ligated rat

Results: We demonstrate 1) That both core tumour and non-tumourderived sequences inhibit the global antiviral effects of IFNa on VSV and EMCV replication in Ratl and HuH7 cells and sensitize Ref cells to transformation, in cooperation with ras oncogene. 2) That tumour and nontumour derived cores show, however, a quite distinct pattern of biological effects on IFNa signaling: inhibition of global antiviral effect of IFNa in tumour-derived expressing cells is much less pronounced, compared with non tumour-derived and reference lb cores.Expression of tumour-derived, and not non-tumour-derived nor reference lb cores, induces transactivafion of 0interferon sensitive response element0 (ISRE); tumour-derived core also increases phosphorylation of Statl and Stat 2, compared with nontumour-derived and reference lb proteins.Tumour-derived core show a markedly increased transforming activity in REF cells, in cooperation with ras oncogene, compared with non-tumour-derived and reference lb cores. Conclusion: Our data 1) support a role for HCV core in the resistance to global antiviral effect of IFNa (despite ISRE activation) and cell transformation in REF cells. 2) demonstrate a distinct pattern of biological effects of tumour and non-tumour-derived cores on interferon signaling and cell transformation, correlated with the hypothesis of selection of core mutants in clonaly expanding liver cells. Data obtained with 3 additional tumour and non-tumour-derived core sequences will be discussed.