In vivo and in silico evaluation of a new nitric oxide donor, S,S'-dinitrosobucillamine.

PURPOSE: In a previous work, we have synthetized a new dinitrosothiol, i.e. S,S'-dinitrosobucillamine BUC(NO)2 combining S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO) in its structure. When exposed to isolated aorta, we observed a 1.5-fold increase of •NO content and a more potent vasorelaxation (1 log higher pD2) compared to NACNO and SNAP alone or combined (Dahboul et al., 2014). In the present study, we analyzed the thermodynamics and kinetics for the release of •NO through computational modeling techniques and correlated it to plasma assays. Then BUC(NO)2 was administered in vivo to rats, assuming it will induce higher and/or longer hypotensive effects than its two constitutive S-mononitrosothiols. METHODS: Free energies for the release of •NO entities have been computed at the density functional theory level assuming an implicit model for the aqueous environment. Degradation products of BUC(NO)2 were evaluated in vitro under heating and oxidizing conditions using HPLC coupled with tandem mass spectrometry (MS/MS). Plasma from rats were spiked with RSNO and kinetics of RSNO degradation was measured using the classical Griess-Saville method. Blood pressure was measured in awake male Wistar rats using telemetry (n = 5, each as its own control, 48 h wash-out periods between subcutaneous injections under transient isoflurane anesthesia, random order: 7 mL/kg vehicle, 3.5, 7, 14 µmol/kg SNAP, NACNO, BUC(NO)2 and an equimolar mixture of SNAP + NACNO in order to mimic the number of •NO contained in BUC(NO)2). Variations of mean (ΔMAP, reflecting arterial dilation) and pulse arterial pressures (ΔPAP, indirectly reflecting venodilation, used to determine effect duration) vs. baseline were recorded for 4 h. RESULTS: Computational modeling highlights the fact that the release of the first •NO radical in BUC(NO)2 requires a free energy which is intermediate between the values obtained for SNAP and NACNO. However, the release of the second •NO radical is significantly favored by the concerted formation of an intramolecular disulfide bond. The corresponding oxidized compound was also characterized as related substance obtained under degradation conditions. The in vitro degradation rate of BUC(NO)2 was significantly greater than for the other RSNO. For equivalent low and medium •NO-load, BUC(NO)2 produced a hypotension identical to NACNO, SNAP and the equimolar mixture of SNAP + NACNO, but its effect was greater at higher doses (-62 ± 8 and -47 ± 14 mmHg, maximum ΔMAP for BUC(NO)2 and SNAP + NACNO, respectively). Its duration of effect on PAP (-50%) lasted from 35 to 95 min, i.e. shorter than for the other RSNO (from 90 to 135 min for the mixture SNAP + NACNO). CONCLUSION: A faster metabolism explains the abilities of BUC(NO)2 to release higher amounts of •NO and to induce larger hypotension but shorter-lasting effects than those induced by the SNAP + NACNO mixture, despite an equivalent •NO-load.

Comparative effects of the angiotensin II receptor blocker, telmisartan, and the angiotensin-converting enzyme inhibitor, ramipril, on cerebrovascular structure in spontaneously hypertensive rats.

OBJECTIVE: Antihypertensive treatment with angiotensin-converting enzyme inhibitors (ACEIs) reverses cerebral arteriolar remodeling, thus restoring dilatation and hence the lower limit of cerebral blood flow (CBF) autoregulation (LLCBF). The objective of this study was to determine whether angiotensin II receptor AT1 blockers (ARBs) produce the same effect. DESIGN: We examined the effects of treatment with an ARB [telmisartan (TEL), 1.93 +/- 0.04 mg/kg per day] or an ACEI [ramipril (RAM), 1.00 +/- 0.02 mg/kg per day] on the cerebral circulation in spontaneously hypertensive rats (SHR). METHODS: Arteriolar pressure and diameter (cranial window) and CBF (laser Doppler) were measured during stepwise hypotensive hemorrhage, before and after deactivation (ethylenediamine tetraacetic acid), in untreated Wistar-Kyoto (WKY) rats and SHR untreated or treated for 3 months with TEL or RAM in the drinking water. RESULTS: Treatment normalized arteriolar internal diameter (SHR, 38 +/- 3 microm; TEL, 52 +/- 2 microm; RAM, 50 +/- 2 microm; WKY, 58 +/- 4 microm), essentially by reversing eutrophic inward remodeling, and the LLCBF (SHR, 80 +/- 11 mmHg; TEL, 60 +/- 4 mmHg; RAM, 71 +/- 6 mmHg; WKY, 57 +/- 5 mmHg). CONCLUSION: The fact that the ARB (TEL) is as effective as an ACEI (RAM) in reversing cerebral arteriolar remodeling suggests that the cerebrovascular AT1 receptor is an underlying mechanism that promotes hypertensive eutrophic inward remodeling.

Captopril improves cerebrovascular structure and function in old hypertensive rats.

We examined the effects of an angiotensin-converting enzyme inhibitor (ACEI), captopril, on cerebral arterioles in young and old spontaneously hypertensive rats (SHR). Animals were anesthetized with sodium pentobarbitone (60 mg kg(-1) day(-1)). We measured cerebral blood flow (CBF, arbitrary units) and cerebral arteriolar internal diameter (ID, mum) prior to and during stepwise hypotension (SH) in 6- (WKY-6) and 15-month-old (WKY-15) Wistar Kyoto rats and in age-matched SHR that were untreated (SHR-6 and SHR-15) or treated for 3 months with captopril (SHR-6C, 105+/-2 mg kg(-1) day(-1) and SHR-15C, 94+/-1 mg kg(-1) day(-1)). ID and cross-sectional area of the vessel wall (CSA) were measured in deactivated (EDTA) cerebral arterioles during a second SH. Captopril decreased the lower limit of CBF autoregulation (61+/-6 in SHR-6C and 51+/-2 in SHR-15C vs 52+/-6 in WKY-6 and 62+/-7 in WKY-15 and 83+/-14 mmHg in SHR-6 and 120+/-19 mmHg in SHR-15; P<0.05) and CSA (510+/-21 in SHR-6C and 585+/-25 in SHR-15C vs 529+/-12 in WKY-6 and 549+/-20 in WKY-15 and 644+/-38 mmHg in SHR-6 and 704+/-38 mmHg in SHR-15; P<0.05). Captopril increased cerebral arteriolar external diameter of SHR (105+/-5 in SHR-6C and 94+/-4 in SHR-15C vs 125+/-8 in WKY-6 and 108+/-3 in WKY-15 and 83+/-2 mmHg in SHR-6 and 80+/-2 mmHg in SHR-15 for a pial arteriolar pressure step of 35-39 mmHg; P<0.05). Captopril attenuated increases in cerebral arteriolar distensibility in young SHR. Thus, ACEIs attenuate eutrophic and hypertrophic inward remodeling of cerebral arterioles in young and old SHR, thus decreasing the lower limit of CBF autoregulation.

Impact of treatment with melatonin on cerebral circulation in old rats.

Melatonin deprival in young rats induces alterations in cerebral arteriolar wall similar to those observed during aging: atrophy and a decrease in distensibility. In this study, we examined the effects of melatonin treatment on cerebral arteriolar structure and distensibility and on the lower limit of cerebral blood flow autoregulation (LLCBF) in old rats. We measured cerebral blood flow (arbitrary unit, laser Doppler, open skull preparation) prior to and during stepwise hypotension (SH) in adult (12/13 months) and old (24/25 months) IcoWI and WAG/Rij male rats. Old rats were untreated or treated for 3 months with melatonin (0.39 (IcoWi) and 0.44 (Wag/Rij) mg kg-1 day-1, drinking water). Stress-strain relationships were determined using cross-sectional area (CSA, microm2, histometry) and values of arteriolar internal diameter (microm) obtained during a second SH following arteriolar deactivation (EDTA, 67 mmol l(-1)). Aging induced (a) atrophy of the arteriolar wall in IcoWI (616+/-20 vs 500+/-27 microm2, P<0.05) but not in WAG/Rij rats (328+/-25 vs 341+/-20 microm2), (b) a decrease in arteriolar wall distensibility and (c) an increase in the LLCBF in both strains (67+/-10 mmHg in 12-month-old vs 95+/-6 mmHg in 24-month-old IcoWi, P<0.05 and 53+/-2 mmHg in 13-month-old vs 67+/-6 mmHg in 25-month-old WAG/Rij). Melatonin treatment induced in IcoWI and WAG/Rij rats (a) hypertrophy of the arteriolar wall (643+/-34 and 435+/-25 microm2, respectively), (b) an increase in arteriolar wall distensibility and (c) a decrease in the LLCBF (64+/-6 and 45+/-4 mmHg, respectively). Melatonin treatment of old rats induced hypertrophy of the arteriolar wall, prevented the age-linked decrease in cerebral arteriolar distensibility and decreased the LLCBF. British Journal of Pharmacology (2004) 141, 399-406. doi:10.1038/sj.bjp.0705629

Differential effects of short-term treatment with two AT1 receptor blockers on diameter of pial arterioles in SHR.

Chronic treatment with angiotensin receptor blockers is largely accepted for protecting cerebral circulation during hypertension, but beneficial effects of short-term treatments are questionable, as highlighted by the recent SCAST trial. We compared the impact of 10 days treatment with candesartan (as SCAST) versus telmisartan (previously described to reverse arteriolar remodeling, chronic treatment) on pial arterioles of spontaneously hypertensive rats (SHR). We explored whether PPAR-gamma agonist activity or AT(1) receptor blockade are involved in their differential effects. In the first study, 4-month-old male SHR were treated with telmisartan (TELMI, 2 mg/kg per day) or candesartan cilexetil (CANDE, 10 mg/kg per day) and compared to vehicle treated SHR and normotensive WKY. In a second study, SHR were treated with CANDE, pioglitazone (a PPAR-gamma agonist, PIO 2.5 mg/kg per day) or CANDE+PIO, compared to TELMI. Internal diameter of pial arterioles (ID, cranial window) was measured at baseline, during hemorrhage-induced hypotension, or following suffusion of Ang II (10(-6) mol/L) or EDTA inactivation of smooth muscle cells (passive ID). PPAR-gamma and eNOS (target gene of PPAR-gamma) mRNA were evaluated in brain microvessels. For similar antihypertensive effects, TELMI (+44% versus SHR), but not CANDE, increased baseline ID. During hemorrhage, ID in TELMI group was similar to WKY, while ID in SHR and CANDE remained lower. In the second study, TELMI (+36%, versus SHR) and CANDE+PIO (+43%) increased baseline ID, but not CANDE or PIO alone. TELMI (-66%) and CANDE+PIO (-69%), but neither CANDE nor PIO alone, decreased Ang II-induced vasoconstriction. CANDE+PIO, but not CANDE, increased passive ID. In both studies, PPAR-gamma and eNOS expressions were higher in TELMI than CANDE. Short-term treatment with TELMI, but not with CANDE, reverses narrowing of pial arteriolar ID in SHR. This may involve PPAR-gamma related mechanisms, since CANDE+PIO treatment induced similar effects, and a better blockade of AT(1) receptors.

Endothelial γ-glutamyltransferase contributes to the vasorelaxant effect of S-nitrosoglutathione in rat aorta.

S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide ((•)NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be catalyzed by γ-glutamyltransferase (GGT). We investigated whether vascular GGT influences the vasorelaxant effect of GSNO in isolated rat aorta. Histochemical localization of GGT and measurement of its activity were performed by using chromogenic substrates in sections and in aorta homogenates, respectively. The role of GGT in GSNO metabolism was evaluated by measuring GSNO consumption rate (absorbance decay at 334 nm), (•)NO release was visualized and quantified with the fluorescent probe 4,5-diaminofluorescein diacetate. The vasorelaxant effect of GSNO was assayed using isolated rat aortic rings (in the presence or absence of endothelium). The role of GGT was assessed by stimulating enzyme activity with cosubstrate glycylglycine, as well as using two independent inhibitors, competitive serine borate complex and non-competitive acivicin. Specific GGT activity was histochemically localized in the endothelium. Consumption of GSNO and release of free (•)NO decreased and increased in presence of serine borate complex and glycylglycine, respectively. In vasorelaxation experiments with endothelium-intact aorta, the half maximal effective concentration of GSNO (EC50 = 3.2 ± 0.5.10(-7) M) increased in the presence of the two distinct GGT inhibitors, serine borate complex (1.6 ± 0.2.10(-6) M) and acivicin (8.3 ± 0.6.10(-7) M), while it decreased with glycylglycine (4.7 ± 0.9.10(-8) M). In endothelium-denuded aorta, EC(50) for GSNO alone increased to 2.3 ± 0.3.10(-6) M, with no change in the presence of serine borate complex. These data demonstrate the important role of endothelial GGT activity in mediating the vasorelaxant effect of GSNO in rat aorta under physiological conditions. Because therapeutic treatments based on GSNO are presently under development, this endothelium-dependent mechanism involved in the vascular effects of GSNO should be taken into account in a pharmacological perspective.

High salt intake abolishes AT(2)-mediated vasodilation of pial arterioles in rats.

BACKGROUND: Angiotensin II (Ang II) induces constriction (AT(1)) and dilation (AT(2) receptors) of cerebral arterioles. High sodium intake induces changes in receptors expression and loss of AT(2)-mediated vasodilation in extracerebral vessels. We investigated whether high salt modifies the AT(2)-mediated response of cerebral arterioles. METHODS: Three-month-old male Wistar rats received drinking water supplemented or not with 1% NaCl. We measured at day 4 or 30 plasma aldosterone concentration, AT receptors expression (brain microvessels, western blot, RT-qPCR), internal diameter of pial arterioles (cranial window) following suffusion with Ang II (10(-6) mol/l, or 10(-8) mol/l + losartan 10(-5) mol/l), serotonin (5-HT, 10(-6) mol/l), sodium nitroprusside (10(-5) mol/l) and adenosine diphosphate (ADP, 10(-4) mol/l). RESULTS: High salt did not modify arterial pressure, baseline arteriolar diameter, vasoconstriction to Ang II or 5-HT, nor vasodilation to SNP. High salt lowered plasma aldosterone concentration (d4 138 ± 71 not significant vs. control 338 ± 73; d30 150 ± 21 P < 0.05 vs. control 517 ± 79 µmol/l). AT receptors mRNA did not change while protein level of AT(2) receptors decreased at d4 (64 ± 9% of control, P < 0.05). AT(2)-mediated vasodilation (control d4; d30 8 ± 2; 5 ± 2%) was abolished at d4 (-2 ± 2%, P < 0.05) and reversed to vasoconstriction at d30 (-7 ± 2%, P < 0.05). ADP-induced vasodilation is abolished at d30 (2 ± 2, P < 0.05 vs. control 19 ± 4%). CONCLUSION: High salt specifically abolishes AT(2)-mediated vasodilation, immediately, via decreased level of AT(2) receptor protein, and after 30 days, in association with abolition of endothelial vasodilation. Such loss of AT(2)-mediated vasodilation may be deleterious in case of stroke.

Effects of suboptimal doses of the AT1 receptor blocker, telmisartan, with the angiotensin-converting enzyme inhibitor, ramipril, on cerebral arterioles in spontaneously hypertensive rat.

OBJECTIVE: Antihypertensive treatment with standard clinical doses of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) reverses cerebral arteriolar remodeling, thus restoring dilatation and the lower limit of cerebral blood flow (CBF) autoregulation (LL CBF AR). In humans, a combination of standard clinical doses of the two drugs does not produce greater protection against stroke than that obtained with single-drug treatments and increases the risk of side-effects. We hypothesized that a combination of suboptimal doses of the ARB, telmisartan (TEL) and of the ACEI, ramipril (RAM), could be a well tolerated and effective treatment of hypertension-induced remodeling of cerebral arterioles. DESIGN: We studied the impact of 3-month oral treatment with TEL (0.5 or 0.8 mg/kg per day) or RAM (0.1 or 0.25mg/ kg per day) alone or in combination (TEL0.8 + RAM0.1 or TEL0.5 + RAM0.25) on the cerebral circulation of the spontaneously hypertensive rats (SHRs). Normotensive Wistar-Kyoto rats (WKYs) were taken as controls. METHODS: Cerebral arteriolar pressure, CBF and internal diameter were measured via an open-skull preparation at baseline and during hypotension before and after deactivation (EDTA). RESULTS: Combinations normalized cerebral arteriolar pressure, whereas drugs alone had no significant impact. TEL0.8 + RAM0.1 showed the greatest effect on arteriolar internal diameter (SHRs 42+/-16, WKYs 59+/-16microm, TEL0.5 + RAM0.25 50+/-6, TEL0.8 + RAM0.1 62+/-18, P<0.05) and normalized LL CBF AR (SHRs 77+/-28, WKYs 53+/-17 mmHg, TEL0.8 + RAM0.1 50+/-10, P<0.05). CONCLUSION: The combination of suboptimal doses of TEL and RAM with an 8 : 1 ratio has the greatest effect on cerebral circulation and could represent well tolerated and efficient treatment of cerebral ischemia and stroke.