Luca Mazzetti

Relaxin up-regulates inducible nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial cells

Relaxin (RLX) is a reproductive hormone with vasodilatatory properties on several organs, including the heart. RLX‐induced vasodilatation appears to depend on the stimulation of endogenous NO production. Here, we investigate whether RLX acts on rat coronary endothelial (RCE) cells in vitro by inducing changes of NO generation and, if so, to clarify the possible mechanism of action. RCE cells were treated for 24 h with vehicle (controls) or RLX, alone or in association with inhibitors of NO synthesis or dexamethasone, which inhibits transcription of NO synthase gene. In some experiments, inactivated RLX was given in the place of authentic RLX. Expression of NO synthase isozymes II and III was analyzed by immunocytochemistry, Western blot, and RT‐PCR. NO production was evaluated by the Griess reaction for nitrite and the NO‐sensitive fluorophore DAF‐2/DA. Agonist‐induced changes of intracellular Ca2+ transient were studied with the Ca2+‐sensitive fluorophore Fura 2‐AM. RLX was found to up regulate NOS II mRNA and protein and to stimulate intrinsic NO generation, likely through the activation of a dexamethasone‐sensitive transcription factor, and to decrease agonist‐induced intracellular Ca2+ transient. Conversely, RLX had negligible effects on NOS III expression. By these biological effects, RLX may afford significant protection against cardiovascular disease.

The ACh‐induced contraction in rat aortas is mediated by the Cys Lt1 receptor via intracellular calcium mobilization in smooth muscle cells

Our previously published data indicate that an endogenously produced 5‐lipoxygenase metabolite can strongly contract isolated endothelium‐preserved rat aortic strips when cyclo‐oxygenase isoenzymes are inhibited. Therefore, we decided to investigate if cysteinyl‐containing leukotrienes (Cys Lts) are involved in this endothelium‐dependent contraction. The isometric contraction of endothelium‐preserved rat aortic strips was recorded in preparations preincubated with 5 μM indomethacin and precontracted with phenylephrine, adjusting resting tension at 0.7 g. Acetylcholine (ACh) contracted control strips. Montelukast and MK‐571, selective type 1 Cys Lts receptor (Cys Lt1) antagonists and the Cys Lt1/Cys Lt2 (type 2 Cys Lts receptor) antagonist BAYu9773 dose‐dependently prevented ACh‐induced contraction, their IC50s being 2.2, 3.1 and 7.9 nM respectively. The leukotriene B4 receptor antagonist U75302 was far less potent (IC50 1.5 μM). In rat aorta smooth muscle cells (RASMs), Western blot analysis showed the presence of Cys Lt1 and Cys Lt2 receptors, the Cys Lt1 receptor being predominantly expressed. In fura‐2 loaded RASMs, LTD4 (0.01–100 nM) and LTC4 (200–800 nM) dose‐dependently increased intracellular calcium concentration ([Ca2+]i). Montelukast (1–100 nM) reduced LTD4‐induced [Ca2+]i increase, its IC50 being approximately 10 nM. BAY u9773 exhibited significantly low effectiveness. LTD4 (10 nM) induced a redistribution of smooth muscle actin fibres throughout the cytoplasm as visualized by confocal microscopy. In conclusion, Cys Lt1 activation by endogenously produced Cys Lts, can contract rat aortas, while Cys Lt2 only marginally influences aortic tone. Intracellularly, this effect is mediated by an increase in [Ca2+]i. Therefore, Cys Lts, by inducing vascular contraction, can contribute to systemic hypertension.

High-yield method for isolation and culture of endothelial cells from rat coronary blood vessels suitable for analysis of intracellular calcium and nitric oxide biosynthetic pathways

We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This method makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.

Mechanical stretch reveals different components of endothelial‐mediated vascular tone in rat aortic strips

Since the role of mechanical stretches in vascular tone regulation is poorly understood, we studied how stretch can influence endothelial tone. Isometric contractions of isolated rat aortic helical strips were recorded. The resting tension was set at 0.7 g, 1.2 g or 2.5 g. Endothelium‐preserved strips were precontracted with either phenylephrine or prostaglandin F2α (PGF2α). In control conditions, acetylcholine (ACh) dose‐dependently relaxed phenylephrine‐precontracted strips independently of resting tension. At 0.7 g resting tension, nitric oxide synthase (NOS) inhibitors did not reduce ACh‐induced relaxation, while either a guanylyl cyclase inhibitor or a NO trapping agent prevented it. At 1.2 g and 2.5 g resting tensions, NOS inhibitors shifted the ACh dose‐response curve to the right. After preincubation with indomethacin (5 μM) or ibuprofen (10 and 100 μM), at 0.7 g and 1.2 g resting tensions, ACh induced an endothelium‐dependent, dose‐dependent contraction. ACh (10−6 M) increased the contraction up to two times greater the phenylephrine‐induced one. Lipoxygenase inhibitors prevented it. At high stretch, the ACh vasorelaxant effect was marginally influenced by cyclooxygenase (COX) inhibition. Similar results were obtained when aortic strips were precontracted with PGF2α. Our data indicate that when resting tension is low, ACh mobilizes a stored NO pool that, synergistically with COX‐derived metabolites, can relax precontracted strips. COX inhibition up‐regulates the lipoxygenase metabolic pathway, accounting for the ACh contractile effect. At an intermediate resting tension, NO production is present, but COX inhibition reveals a lipoxygenase‐dependent, ACh‐induced contraction. At high resting tension, NO synthesis predominates and COX metabolites influence ACh‐induced relaxation marginally.

Effects of Relaxin on Vascular Smooth Muscle and Endothelial Cells in Normotensive and Hypertensive Rats

Abstract: We tested the effects of relaxin on [Ca2+]i response to angiotensin II in smooth muscle (vSMC) and endothelial cells isolated from hypertensive (SHR) and normotensive (WKY) rats. Relaxin markedly reduced the [Ca2+]i response of vSMCs from WKY, but not from SHR rats. Western blots showed that cGMP‐dependent protein kinase G was reduced in vSMCs from SHR as compared with WKY rats. Relaxin also blunted the [Ca2+]i response in endothelial cells from WKY, but not from SHR rats. However, in endothelial cells from SHR and WKY rats, protein kinase G was nearly unexpressed, thus accounting for an alternative pathway of the intracellular response to nitric oxide and relaxin. Hence, vSMCs and endothelial cells in SHR rats show a deficiency response to nitric oxide that may render them insensitive to relaxin.

Guanosine 3': 5'-cyclic monophosphate-dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats.

We investigated the effect of the NO‐donor S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura‐2‐loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells. In WKY cardiomyocytes, 1 – 200 μM SNAP dose‐dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level. In control conditions, the ΔF/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 – 100 μM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10−6 – 10−8 M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP. In WKY and SHR cardiomyocytes, 10−8 M isoprenaline similarly increased ΔF/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT‐5823. Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations. We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP‐dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.

Evaluation of the effects of a novel carbon monoxide releasing molecule (CORM-3) in an in vitro model of cardiovascular inflammation.

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Lack of nitric oxide‐ and guanosine 3′:5′‐cyclic monophosphate‐dependent regulation of α‐thrombin‐induced calcium transient in endothelial cells of spontaneously hypertensive rat hearts

While the expression and/or activity of endothelial nitric oxide synthase (eNOS) has been characterized in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rat (WKY) hearts, in coronary endothelial cells (ECs) from both strains, the effect of NO on intracellular calcium concentration ([Ca2+]i) is still unknown. Coronary microvascular ECs were isolated from SHR and WKY and characterized. Immunocytochemistry and Western blot analysis showed that eNOS was similarly expressed in ECs from both strains. Measuring [Ca2+]i by imaging analysis of fura‐2‐loaded cells, we demonstrated that α‐thrombin (3−180 U l−1) induced a superimposable dose‐dependent calcium transient in ECs from both strains. In WKY ECs, S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) dose‐dependently (10–100 μM) and 0.1 μM atrial natriuretic factor (ANF) reduced the maximum and the decay time of α‐thrombin‐induced calcium transient. The inhibitory effects of SNAP and ANF were prevented by blocking cyclic GMP‐dependent protein kinase. Non selective eNOS inhibitors prolonged the decay time of α‐thrombin‐induced calcium transient, while the selective inducible NOS inhibitor 1400 W was ineffective. SNAP (100 μM) and 0.1 μM ANF increased cyclic GMP content up to 22.9 and 42.3 fold respectively. In SHR ECs, α‐thrombin‐induced calcium transient was not modified by SNAP, ANF or eNOS inhibition. SNAP (100 μM) and 0.1 μM ANF increased cyclic GMP content up to 9.3 and 51 fold respectively. In WKY ECs, SNAP dose‐dependently (10–100 μM) reduced also bradykinin‐induced calcium transient, while in SHR ECs was ineffective. We concluded that in SHR ECs, the cyclic GMP‐dependent regulation of calcium transient is lost.

Altered nitric oxide calcium responsiveness of aortic smooth muscle cells in spontaneously hypertensive rats depends on low expression of cyclic guanosine monophosphate-dependent protein kinase type I

Objectives The nitric oxide/cyclic guanosine monophosphate (GMP)/cyclic GMP-dependent protein kinase type I (cGKI) pathway has been extensively investigated in the spontaneously hypertensive rat (SHR) as a possible pathogenetic factor. Therefore, we investigated the role of nitric oxide/cGKI on intracellular calcium dynamics ([Ca2+]i) of aortic smooth muscle cells isolated from control normotensive Wistar Kyoto rats (WKY) and SHR. Methods Rat aortic smooth muscle cells (RASMCs) were obtained from 12 to 16-week-old WKY and SHR. [Ca2+]i dynamics were monitored by imaging analysis of fura-2-loaded RASMCs. cGKI mRNA and cGKI protein expression were evaluated by reverse transcription-PCR and western blot. Plasmids codifying for enhanced green fluorescent protein (EGFP) or cGKIα–EGFP were transfected on SHR RASMCs. Results Angiotensin II similarly increased [Ca2+]i in WKY and SHR RASMCs. In WKY RASMCs, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 1–100 μmol/l) reduced the decay time of angiotensin II-induced [Ca2+]i transient. On the contrary, in SHR cells, SNAP was ineffective. Dibutyryl cyclic GMP (1–100 nmol/l), a membrane-permeable cyclic GMP analogue, behaved similarly to SNAP. In naive SHR RASMCs, cGKI mRNA and cGKI protein were low or absent. After transfection of a plasmid encoding for cGKIα–EGFP, the [Ca2+]i dynamic of SHR-transfected cells regained sensitivity to the nitric oxide/cyclic GMP pathway. Conclusion The low expression of cGKI determines the lack of nitric oxide/cyclic GMP-dependent regulation on [Ca2+]i transient in SHR RASMCs. This alteration may contribute to the development of hypertension and explain suboptimal responses to nitroglycerin and other nitric oxide-releasing molecules in patients.

Restoring nitric oxide cytosolic calcium regulation by cyclic guanosine monophosphate protein kinase I alpha transfection in coronary endothelial cells of spontaneously hypertensive rats.

In microcoronary endothelial cells (RCEs) from spontaneously hypertensive rats (SHR), the nitric oxide (NO)/cyclic guanosine monophosphate (GMP)-dependent proteinkinase I (cGKI) pathway cannot regulate the cytosolic calcium ([Ca2+]i) dynamic as in RCEs from Wistar Kyoto rats (WKY). We investigated the altered downstream NO target in SHR cells and, since cGKI expression was low, whether the re-expression of cGKIα in SHR RCEs could restore NO calcium responsiveness. We measured [Ca2+]i dynamic by fura-2 imaging analysis and the cGKI level by RT-PCR and Western blot in SHR and WKY RCEs. Plasmids encoding for enhanced green fluorescence protein or cGKIα-enhanced green fluorescence protein were transiently transfected in SHR RCEs, and [Ca2+]i was evaluated. Angiotensin-II (AT-II) increased [Ca2+]i in a concentration-dependent way in both strains. Whereas in WKY, endogenously produced NO and cyclic GMP analog decreased the AT-II-induced [Ca2+]i transient, they were ineffective in SHR RCEs. The cGKI level was low in SHR cells. However, after cGKIα re-expression, endogenous NO decreased the AT-II-induced [Ca2+]i transient, while endothelial NO synthase and cGKI inhibition prevented it. The low expression of cGKI in SHR accounts for the absent regulation of the agonist-induced [Ca2+]i transient by the NO/cyclic GMP pathway. Studies on cGKI in humans could contribute to a better understanding of cardiovascular pathologies.