Endothelin ETB Receptor Antagonist Research Articles

Angiopoietin-1/Tie-2 signal after focal traumatic brain injury is potentiated by BQ788, an ETB receptor antagonist, in the mouse cerebrum: Involvement in recovery of blood-brain barrier function.

Angiopoietin-1, an angiogenic factor, stabilizes brain microvessels through Tie-2 receptor tyrosine kinase. In traumatic brain injury, blood-brain barrier (BBB) disruption is an aggravating factor that induces brain edema and neuroinflammation. We previously showed that BQ788, an endothelin ETB receptor antagonist, promoted recovery of BBB function after lateral fluid percussion injury (FPI) in mice. To clarify the mechanisms underlying BBB recovery mediated by BQ788, we examined the involvements of the angiopoietin-1/Tie-2 signal. When angiopoietin-1 production and Tie-2 phosphorylation were assayed by quantitative reverse transcription polymerase chain reaction and western blotting, increased angiopoietin-1 production and Tie-2 phosphorylation were observed in 7-10days after FPI in the mouse cerebrum, whereas no significant effects were obtained at 5days. When BQ788 (15nmol/day, i.c.v.) were administered in 2-5days after FPI, increased angiopoietin-1 production and Tie-2 phosphorylation were observed. Immunohistochemical observations showed that brain microvessels and astrocytes contained angiopoietin-1 after FPI, and brain microvessels also contained phosphorylated Tie-2. Treatment with endothelin-1 (100nM) decreased angiopoietin-1 production in cultured astrocytes and the effect was inhibited by BQ788 (1μM). Five days after FPI, increased extravasation of Evans blue dye accompanied by reduction in claudin-5, occludin, and zonula occludens-1 proteins were observed in mouse cerebrum while these effects of FPI were reduced by BQ788 and exogenous angiopoietin-1 (1μg/day, i.c.v.). The effects of BQ788 were inhibited by co-administration of a Tie-2 kinase inhibitor (40nmol/day, i.c.v.). These results suggest that BQ788 administration after traumatic brain injury promotes recovery of BBB function through activation of the angiopoietin-1/Tie-2 signal.

Blockade of endothelin receptors reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial carcinoma induced pain

Pain reported by patients with head and neck cancer is characterized as persistent pain with mechanical allodynia. Pain management is inadequate for many patients, highlighting the need for improved therapies. We examined the hypothesis that the mixed endothelin ETA and ETB receptor antagonist, bosentan, reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial cancer pain. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rat's right vibrissal pad. Tumor-bearing rats developed heat and tactile hypersensitivity along with increased spontaneous grooming behavior. Systemic morphine (2.5mg/kg, s.c.) blocked tumor-induced thermal and tactile hypersensitivity, with a lower dose (0.625mg/kg, s.c.) effective only against thermal hypersensitivity. Systemic bosentan blocked tumor-induced thermal hypersensitivity only at a high (300mg/kg, p.o.) dose, but failed to modify tactile hypersensitivity. Co-administration of the low doses of bosentan and morphine resulted in improved reduction of the tumor-induced heat and tactile hypersensitivity compared to either dose alone. Bosentan (100mg/kg, p.o.) reduced spontaneous grooming and induced conditioned place preference (CPP) selectively in tumor-bearing rats, suggesting that bosentan reduces tumor-induced ongoing pain at a lower dose than required to block tumor-induced hypersensitivity. This study provides evidence that endothelins may mediate tumor-induced ongoing pain and thermal hypersensitivity. In addition, bosentan enhanced morphine's effects on blocking tumor-induced heat and tactile hypersensitivity indicating that endothelin antagonists may be beneficial therapeutic targets that can be used to manage cancer-induced facial pain with opioid-sparing effects.

The new endothelin receptor antagonist macitentan: Prospects for therapy of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a clinical group of severe and rare diseases with similar morphological, hemodynamic, and therapeutic characteristics. One of the novel drugs to treat PAH is macitentan, a new double endothelin ETA and ETB receptor antagonist that is characterized by special physicochemical properties, ensuring the penetration of the drug into tissues and its improved receptor-binding properties. The SERAPHIN trial has demonstrated that therapy with macitentan 10 mg versus placebo statistically significantly reduces the risk of poor outcomes and death by 45%. The treatment with macitentan 10 is observed to be highly effective regardless of the presence/absence of basic PAH-specific therapy. The drug considerably improves clinically important outcomes, including 6-minute walk distance and WHO functional class. Macitentan exerts a steady-state therapeutic effect, by improving pulmonary hemodynamics. Macitentan 10 mg statistically significantly reduces the risk of PAH, frequency of its related hospitalizations, and the number of days spent in hospital. The drug has a favorable safety profile; its most common side effects are headache, nasopharyngitis, and anemia. Macitentan is an effective first-line drug to improve long-term outcomes in patients with newly and previously diagnosed PAH.

Celecoxib offsets the negative renal influences of cyclosporine via modulation of the TGF-β1/IL-2/COX-2/endothelin ETB receptor cascade

Endothelin (ET) signaling provokes nephrotoxicity induced by the immunosuppressant drug cyclosporine A (CSA). We tested the hypotheses that (i): celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, counterbalances renal derangements caused by CSA in rats and (ii) the COX-2/endothelin ETB receptor signaling mediates the CSA-celecoxib interaction. Ten-day treatment with CSA (20mg/kg/day) significantly increased biochemical indices of renal function (serum urea, creatinine), inflammation (interleukin-2, IL-2) and fibrosis (transforming growth factor-β1, TGF-β1). Histologically, CSA caused renal tubular atrophy along with interstitial fibrosis. These detrimental renal effects of CSA were largely reduced in rats treated concurrently with celecoxib (10mg/kg/day). We also report that cortical glomerular and medullary tubular protein expressions of COX-2 and ETB receptors were reduced by CSA and restored to near-control values in rats treated simultaneously with celecoxib. The importance of ETB receptors in renal control and in the CSA-celecoxib interaction was further verified by the findings (i) most of the adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ETB receptor antagonist BQ788 (0.1mg/kg/day, 10 days), and (ii) the BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, the data suggest that the facilitation of the interplay between the TGF-β1/IL-2/COX-2 pathway and the endothelin ETB receptors constitutes the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in rats.

Celecoxib Offsets The Negative Renal Influences of Cyclosporine Via COX‐2/Endothelin ETB Receptor crosstalk

We tested the hypotheses that (i) celecoxib, a selective COX‐2 inhibitor, counterbalances renal derangements caused by cyclosporine A (CSA) in male rats, and (ii) the endothelin ETB receptor mediates the CSA‐celecoxib interactions. Ten‐day treatment with CSA (20 mg/kg/day) significantly increased biochemical indices of renal function (serum urea and creatinine) and inflammatory cytokines (renal transforming growth factor‐β and interleukin‐2). CSA also caused renal tubular atrophy and congestion in renal arterioles along with perivascular and interstitial fibrosis. These detrimental renal effects of CSA were dramatically reduced in rats treated concurrently with celecoxib (10 mg/kg/day). Moreover, immunohistochemical analyses showed that glomerular and tubular protein expressions of COX‐2 and ETB were reduced by CSA and restored to near‐control values in rats treated simultaneously with celecoxib. The adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ETB receptor antagonist BQ788 (0.1 mg/kg/day, 10 days). The BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, facilitation of the interplay between COX‐2 and endothelin ETB receptors is the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in male rats.

Endothelin A receptor antagonist modulates lymphocyte and eosinophil infiltration, hyperreactivity and mucus in murine asthma

Levels of endothelins are particularly high in the lung, and there is evidence that these peptides are involved in asthma. Asthma is a chronic inflammatory disease associated with lymphocyte infiltration. In the present study, we used a murine model of asthma to investigate the role of endothelins in lymphocyte and eosinophil infiltration into the airway hyperreactivity and mucus secretion. Sensitized C57Bl/6 mice were treated with endothelin ETA receptor antagonist (BQ123) or endothelin ETB receptor antagonist (BQ788) 30 min before an antigen aerosol challenge. After 24 h, dose response curves to methacholine were performed in isolated lungs, FACS analysis of lymphocytes and eosinophil counts were performed in bronchoalveolar lavage fluid and mucus index was determined by histopathology. In sensitized and antigen-challenged mice there is a marked increase in the T CD4+, T CD8+, B220+, Tγδ+ and NK1.1+ lymphocyte subsets. Treatment with BQ123 further increased these cell populations. The number of eosinophils, airway hyperreactivity and mucus were all reduced by BQ123 treatment. The BQ 788 had no significant effect on the parameters analyzed. Treatment with BQ123 reduced the endothelin concentration in lung homogenates, suggesting that endothelins exert a positive feedback on their synthesis. We show here that in murine asthma the ETA receptor antagonist up-regulates lymphocyte infiltration and reduces eosinophils, hyperreactivity and mucus.

Selective Endothelin ETB Receptor Antagonist Improves Left Ventricular Function but Exaggerates Degeneration of Cardiomyocytes in J2N-k Hamsters

Endothelin-1 (ET-1) receptor antagonist is expected to improve the prognosis of patients with heart failure, but the role of the ET(B) receptor in cardiac function and structure is complicated. In the present study the NADPH diaphorase activity and ET-1 content in the failing heart treated with ET(A) or ET(B) receptor antagonist were evaluated in a model of dilated cardiomyopathy. Selective ET(A) receptor antagonist, ABT-627 (10 mg/kg per day), or selective ET(B) antagonist, A-192621 (30 mg/kg per day), was given to 22-week-old J2N-k cardiomyopathic hamsters for 8 weeks. The effects of ABT-627 and A-192621 on cardiac function, left ventricular (LV) histology, ET-1 content and NADPH diaphorase activity in the LV were evaluated. Treatment with ABT-627, but not A-192621, significantly decreased ET-1 content and NADPH diaphorase activity. Although the improvement of LV function was modest, ABT-627 prevented tissue damage in J2N-k hamsters. In contrast, A-192621 worsened the degeneration of cardiomyocytes despite improving hemodynamic parameters. Selective ET(A) antagonist, but not ET(B) antagonist, reduced the ET-1 content as well as the NADPH diaphorase activity, and preserved the fine structure of LV myocardium in cardiomyopathic hamsters. Long-term blockade of ET(B) receptor might worsen the degeneration of cardiomyocytes through the ET-1/ET(A) system even if LV function could be improved.

Dual effects of endothelin-1 (1-31): induction of mesangial cell migration and facilitation of monocyte recruitment through monocyte chemoattractant protein-1 production by mesangial cells.

We previously found that human chymase selectively cleaves big endothelin-1 (ET-1) at the Tyr31-Gly32 bond and produces 31-amino acid endothelins, ET-1 (1-31), without any further degradation products. In this study, we investigated the effect of ET-1 (1-31) on the migration of cultured rat mesangial cells (RMCs) and on cells of the human monocytic cell line, THP-1. In addition, we examined the interaction between RMCs and THP-1 cells using conditioned media from ET-1 (1-31)-stimulated RMCs. ET-1 (1-31) caused an increase in RMC migration in a concentration-dependent manner, and the degree of increase was similar to those by ET-1 and angiotensin II (All). The ET-1 (1-31)-induced increase in RMC migration was inhibited by BQ123, an endothelin ETA receptor antagonist, but not by BQ788, an endothelin ETB receptor antagonist. ET-1 (1-31) alone did not cause significant migration of THP-1 cells. However, significant recruitment of THP-1 cells was observed with conditioned media taken from ET-1 (1-31)-stimulated RMCs. The conditioned media-induced migration of THP-1 cells was inhibited by BQ123, but not by BQ788. Western blotting analysis of the lysate of RMCs revealed that the expression of monocyte chemoattractant protein-1 (MCP-1) protein in RMCs was increased by treatment with ET-1 (1-31). The addition of neutralizing antibody for MCP-1 to the medium inhibited the migration of THP-1 cells induced by conditioned media from ET-1 (1-31)-stimulated RMCs. These findings suggest that ET-1 (1-31) play a role in glomerulonephritis (GN) via dual effects that directly cause the migration of mesangial cells (MCs) and may be responsible for the recruitment of mononuclear cells mediated through the activation of MCs. Since human chymase has been reported to be involved in glomerular disease, ET-1 (1-31) may be among the mediators.

Impaired potentiation by endothelin-1 and vasopressin of sympathetic contraction in tail artery from hypertensive rats.

To analyse the effects of endothelin-1 and vasopressin on the sympathetic vasoconstriction during hypertension. Electrical field stimulation (4 Hz) was applied to isolated, 2 mm segments of the tail artery from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats prepared for isometric tension recording. The contraction to electrical stimulation was potentiated by endothelin-1 (10(-10)-10(-8) M) in arteries from WKY but not from SHR, and by vasopressin (10(-12)-10(-10) M) more markedly in arteries from WKY than from SHR. The potentiation by endothelin-1 was reduced more markedly by the antagonist of endothelin ETA receptors BQ-123 (10(-5) M) than by the endothelin ETB receptor antagonist BQ-788 (10(-5) M). The potentiation by vasopressin was reduced by the antagonist of vasopressin V1 receptors d(CH2)5Tyr(Me)AVP (10(-7) M), but not by the vasopressin V2 receptor antagonist d(CH2)5D-Ile2, Ile4AVP (10(-7) M). The blocker of L-type calcium channels verapamil (10(-5) M) reduced the potentiation by both endothelin-1 and vasopressin in arteries from WKY rats, and increased the potentiation by vasopressin in arteries from SHR. Noradrenaline (10(-8)-10(-4) M) contraction was not modified by endothelin-1 (3 x 10(-9) M) or vasopressin (3 x 10(-11) M), and contraction to endothelin-1 (10(-9)-10(-7) M) and vasopressin (10(-10)-10(-7) M) was lower in arteries from SHR than from WKY rats. (1) The potentiation by endothelin-1 and vasopressin of the sympathetic vasoconstriction, probably due to increased release of noradrenaline, is impaired during hypertension, and (2) this potentiation is mediated mainly by endothelin ETA receptors, and by vasopressin V1 receptors, in both WKY and SHR, and for both peptides it is mediated by L-type calcium channels in arteries from normotensive but not in those from hypertensive animals.

BQ788, an endothelin ET(B) receptor antagonist, attenuates stab wound injury-induced reactive astrocytes in rat brain.

Endothelins (ETs) are suggested to be involved in pathological or pathophysiological responses on brain injuries. In the present study, an involvement of ETs on activation of astrocytes in vivo was examined by using selective endothelin receptor antagonists. A stab wound injury on rat cerebral cortex increased immunoreactive ET-1 at the injured site. GFAP-positive [GFAP(+)] and vimentin-positive [Vim(+)] cells appeared at the injured site in 1 day to 2 weeks after the injury. A continuous infusion of BQ788, a selective ETB receptor antagonist, into cerebral ventricle (23 nmole/day) attenuated increase in the numbers of GFAP(+) and Vim(+) cells after the injury. FR139317, a selective ETA antagonist (23 nmole/day), slightly decreased the number of Vim(+) cells but not that of GFAP(+) cells. Increase in the number of microglia/macrophages by a stab wound injury, which was determined by Griffonia simplicifolia isolectin B4 staining, was not affected by BQ788 and FR139317. These results suggest that activation of glial ETB receptors is one of the signal cascades leading to reactive astrocytes on brain injuries.

Endothelin-1 enhances vascular cell adhesion molecule-1 expression in tumor necrosis factor alpha-stimulated vascular endothelial cells.

Vascular cell adhesion molecule-1 (VCAM-1) is a mononuclear leukocyte-selective adhesion molecule that is expressed in human vascular endothelial cells at sites of local inflammation. It participates in local endothelial-monocyte interactions during the initiation of atherosclerosis. In the present study, endothelin alone did not induce the surface expression and mRNA accumulation of VCAM-1 in human vascular endothelial cells, but inhibition of endogenous nitric oxide (NO) by N(G)-monomethyl-L-arginine enhanced the surface expression and mRNA accumulation of VCAM-1 stimulated by endothelin-1. It is conceivable that in human vascular endothelial cells, stimulation of an endothelin receptor results in the production of nitric oxide (NO), suppressing the expression of VCAM-1. Endothelin-1 enhanced the surface expression and mRNA accumulation of VCAM-1 in cells treated with tumor necrosis factor alpha (TNF-alpha). The enhancement by endothelin-1 may be explained by the inhibitory effect of TNF-alpha on endothelin-induced NO production. Pretreatment with BQ788 (an endothelin ET(B) receptor antagonist) or inhibitors of nuclear factor kappa B (NF-kappaB) activation completely diminished the synergistic enhancement of VCAM-1 expression by endothelin-1 in TNF-alpha-stimulated vascular endothelial cells, both at the protein and mRNA levels. These findings suggest that the synergistic enhancement of VCAM-1 expression by TNF-alpha and endothelin ET(B) receptor stimulation may be augmented by the induction of NF-kappaB binding activity in human vascular endothelial cells.

Renal effects of endothelin in anesthetized rabbits

Intrarenal arterial infusion of endothelin-1 (1, 3 and 10 ng/kg per min) reduced renal blood flow, urine flow rate and urinary Na+ excretion without affecting fractional Na+ excretion in anesthetized rabbits. An endothelin ETA receptor antagonist (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methyl-pentanoyl]amino-3-[3-(1-methyl-1H-indolyl)]propionyl]amino-3-(2-pyridyl)propionic acid (FR139317, 1 μg/kg per min) attenuated the endothelin-1 (1 ng/kg per min)-induced renal responses. An endothelin ETB receptor antagonist N-cis 2,6-dimetylpiperidinocarbonyl-l-γ-metylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine (BQ-788, 1 μg/kg per min) potentiated the endothelin-1-induced changes in renal blood flow, urine flow rate and urinary Na+ excretion. A nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME, 50 μg/kg per min) also potentiated the endothelin-1-induced reductions in urine flow rate and urinary Na+ excretion but not the reduction in renal blood flow. Endothelin-1 reduced fractional Na+ excretion in the presence of BQ-788 or l-NAME. A spontaneous NO donor 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (30 ng/kg per min) slightly attenuated the antinatriuresis but not the vasoconstriction induced by endothelin-1. These results suggest that in the rabbit kidney in vivo endothelin ETA receptors mediate endothelin-1-evoked vasoconstriction and tubular Na+ reabsorption, that the concomitant stimulation of endothelin ETB receptors by endothelin-1 counteracts both the ETA receptor-mediated vascular and tubular actions, and that the tubular action, but not the vascular action, of endothelin-1 is also susceptible to changes in renal NO level.

Localization of endothelin ETB receptors on the myenteric plexus of guinea-pig ileum and the receptor-mediated release of acetylcholine.

1. The type of endothelin (ET) receptor located on the myenteric neurones of guinea-pig ileum was determined by receptor autoradiography and function of the receptor was examined by release experiments of acetylcholine (ACh) from the longitudinal muscle myenteric plexus (LM-MP) preparations. 2. Specific [125I]-ET-1 binding sites were distributed in muscle layers, myenteric and submucous plexuses, and mucosa layers. High-grain densities were detected in both myenteric and submucous plexuses. 3. Binding in the myenteric plexus was abolished by incubation with either IRL 1620 (endothelin ETB receptor agonist) or BQ 788 (endothelin ETB receptor antagonist), but not with BQ 123 (endothelin ETA receptor antagonist). The [125I]-IRL 1620 binding sites were evident in the myenteric plexus. Thus, the endothelin receptor located on the myenteric neurones is of the ETB type. 4. ET-1 (10(-10)-3 x 10(-8) M) and ET-3 (10(-10)-3 x 10(-8) M) evoked 3H outflow from LM-MP preparations of ileum preloaded with [3H]-choline, in a concentration-dependent manner. There was no significant difference between maximum amounts of ET-1-evoked and ET-3-evoked 3H outflow. 5. ET-1 and ET-3 evoked outflow of 3H was BQ 788-sensitive, but BQ 123-insensitive. Both evoked outflows of 3H were Ca(2+)-dependent and tetrodotoxin-sensitive. 6. These results indicate that the endothelin ETB receptor is located on the enteric cholinergic neurones and that stimulation evokes the release of ACh.

Selective Blockade of Endothelin Receptor Subtypes on Systemic and Renal Vascular Responses to Endothelin-1 and IRL1620, a Selective Endothelin ETB-Receptor Agonist, in Anesthetized Rats

By using BQ-788 as a selective endothelin ETB-receptor antagonist and FR139317 as a selective endothelin ETA-receptor antagonist, we have characterized the receptor subtypes mediating the systemic and renal vascular effects of endothelin-1 and IRL1620, a selective endothelin ETB-receptor agonist (succinyl-[Glu9,Ala11,5]-endothelin-1(8–21)), in anesthetized rats. Bolus intravenous injection of endothelin-1 (0.5 nmol/kg) and IRL1620 (1.65 nmol/kg) produced a transient fall in systemic blood pressure followed by a sustained increase. The initial fall in blood pressure observed after endothelin-1 and IRL1620 administration was completely blocked by BQ-788 (0.5 μmol/kg, i.v.), whereas the pressor response was blocked by FR139317 (0.8 //mol/kg, i.v.). Renal blood flow was decreased and calculated renal vascular resistance was dramatically increased by endothelin-1 and IRL1620. The reduction of renal blood flow by endothelin-1 was significantly suppressed by FR139317 but potentiated by BQ-788. Both BQ-788 and FR139317 partially blocked the renal vasoconstriction by IRL1620. Pretreatment by BQ-788 itself decreased renal blood flow by 14.1%. These results indicate that the systemic depressor responses induced by endothelin-1 and IRL1620 are mediated through the endothelin ETB-receptor, and the pressor responses are mediated through the endothelin ETA-receptor. In the renal vasculature of anesthetized rats, it is suggested that vasoconstriction is mediated through both endothelin ETA- and ETB-receptors and that endothelin ETB-receptors may be also involved in vasodilating responses to endothelin peptides.

Endothelin ETB receptor antagonist, RES-701-1: effects on isolated blood vessels and small intestine.

RES-701-1 (cyclic (Gly1-Asp9)(Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Phe- Phe-Asn-Tyr-Tyr-Trp)), a peptide isolated from Streptomyces sp., has been reported to inhibit the endothelin ETB receptor. We examined the effects of this peptide on the blood vessels and the small intestine. In isolated rat aorta without endothelium, 10 microM RES-701-1 did not affect the resting tone, nor did it attenuate the contractions induced by endothelin-1, endothelin-3 or norepinephrine. In the aorta with endothelium, 3 microM RES-701-1 shifted the concentration-response curves for the contractile effects of endothelin-1 and endothelin-3 to the left. Removal of endothelium showed a similar effect to 3 microM RES-701-1. In the norepinephrine-stimulated aorta, endothelium-dependent relaxation induced by endothelin-3 was antagonized by 0.3-10 microM RES-701-1 in a concentration-dependent manner. In the guinea pig ileum stimulated by carbachol, endothelin-3 induced a transient relaxation followed by sustained relaxation. RES-101-1 (3 microM) selectively inhibited the transient relaxation. Since it has been shown that the contractile effects of endothelins in the aorta are mediated by the endothelin ETA receptor whereas the endothelium-dependent relaxation and the ileal relaxation are mediated by the endothelin ETB receptor, it is suggested that RES-701-1 is a selective antagonist against the endothelin ETB receptor.

O-338 - Effects of a novel endothelin ETB receptor antagonist, RES-701-1, on isolated blood vessels.

O-338 - Effects of a novel endothelin ETB receptor antagonist, RES-701-1, on isolated blood vessels.