Chronotropic Effect Of Isoprenaline Research Articles

Synthesis, pharmacological activity, and chromatographic enantioseparation of new heterocyclic compounds of the aryloxyaminopropanol type derived from 4-hydroxyphenylalkanones

In the paper, a series of six pharmacologically active compounds (β-adrenolytics) derived from 4-hydroxyphenylethanone and 4-hydroxyphenylpropan-1-one are reported. The compounds incorporate pyrrolidin-1-yl and 4-methylpiperazin-1-yl substituents in the hydrophilic part of the molecule and ethoxymethyl and methoxyethoxymethyl side chains on the aromatic ring in the lipophilic moiety. They were prepared by a four-step synthesis from 4-hydroxyalkanones via chloromethyl, alkoxymethyl, and oxirane intermediates. The purity of the target compounds was checked by TLC and their structures were confirmed by the interpretation of the IR, UV, 1H NMR, and 13C NMR spectra. The pharmacological evaluation of the obtained compounds confirmed their vasodilatory and specific antiisoprenaline activities. All evaluated compounds at conc. 10−6 mol dm−3 inhibited vasoconstrictory effect of phenylephrine (8.22–33.7%) on isolated rat aorta. The ability to inhibit positive chronotropic effect of isoprenaline was observed on isolated spontaneously beating rat’s atria after pre-treatment with the evaluated compounds at conc. 10−7 and 10−6 mol dm−3. The calculated pA2 values of specific antagonistic effect against isoprenaline, related to their apparent β-adrenolytic activity, ranged between 6.54 and 7.57. The value for the standard compound carvedilol was 8.15 ± 0.22. The majority of the evaluated compounds at conc. 10−6–10−7 mol dm−3 also showed negative chronotropic effect on the basic heart rate of atria. Enantioseparation of the prepared compounds was performed by chiral HPLC on an amylose tris(3,5-dimethylphenylcarbamate) column (Chiralpak AD) and a native teicoplanin column (Chirobiotic T). The chromatographic characteristics as retention, separation, and resolution factors were reported.

Modeling the Chronotropic Effect of Isoprenaline on Rabbit Sinoatrial Node

Introduction: β-adrenergic stimulation increases the heart rate by accelerating the electrical activity of the pacemaker of the heart, the sinoatrial node (SAN). Ionic mechanisms underlying the actions of β-adrenergic stimulation are not yet fully understood. Isoprenaline (ISO), a β-adrenoceptor agonist, shifts voltage-dependent If activation to more positive potentials resulting in an increase of If, which has been suggested to be the main mechanism underlying the effect of β-adrenergic stimulation. However, ISO has been found to increase the firing rate of rabbit SAN cells when If is blocked. ISO also increases ICaL, Ist, IKr, and IKs; and shifts the activation of IKr to more negative potentials and increases the rate of its deactivation. ISO has also been reported to increase the intracellular Ca2+ transient, which can contribute to chronotropy by modulating the “Ca2+ clock.” The aim of this study was to analyze the ionic mechanisms underlying the positive chronotropy of β-adrenergic stimulation using two distinct and well established computational models of the electrical activity of rabbit SAN cells. Methods and results: We modified the Boyett et al. (2001) and Kurata et al. (2008) models of electrical activity for the central and peripheral rabbit SAN cells by incorporating equations for the known dose-dependent actions of ISO on various ionic channel currents (ICaL, Ist, IKr, and IKs), kinetics of IKr and If, and the intracellular Ca2+ transient. These equations were constructed from experimental data. To investigate the ionic basis of the effects of ISO, we simulated the chronotropic effect of a range of ISO concentrations when ISO exerted all its actions or just a subset of them. Conclusion: In both the Boyett et al. and Kurata et al. SAN models, the chronotropic effect of ISO was found to result from an integrated action of ISO on ICaL, If, Ist, IKr, and IKs, among which an increase in the rate of deactivation of IKr plays a prominent role, though the effect of ISO on If and [Ca2+]i also plays a role.

The effects of acute reserpine administration on the sensitivity of the isolated pacemaker from rat heart to isoprenaline and noradrenaline.

The effects of reserpine on the sensitivity of the isolated pacemaker from rat heart to the chronotropic effect of isoprenaline and noradrenaline were studied. A single large dose of reserpine (2.5 mg kg-1) administered to rats 24 h before killing induces supersensitivity of the isolated pacemaker to isoprenaline, leaving unaltered the responsiveness of the pacemaker to noradrenaline. Reserpine at the dose of 1.0 mg kg-1 did not alter the sensitivity of the pacemaker to the catecholamines. Only the larger dose of reserpine raised the corticosterone plasma level. It is possible that a corticosterone-mediated inhibition of the extraneuronal uptake process is responsible for the supersensitivity to isoprenaline. Large doses of reserpine should not be used in experiments aimed to study cardiac sensitivity to isoprenaline or extraneuronal uptake and metabolism of the catecholamine.

RECRUITMENT OF FUNCTIONALLY ACTIVE HEART β2-ADRENOCEPTORS IN THE INITIAL PHASE OF ENDOTOXIC SHOCK IN PITHED RATS

A supersensitivity of the beta-adrenoceptor-mediated chronotropic response has been demonstrated in atria isolated from rats subjected to septic shock. Our study was undertaken to investigate whether bacterial endotoxin/LPS affects the increase in heart rate induced by beta-adrenoceptor agonists in the rat also in vivo. In pithed and vagotomized rats, the nonselective beta-adrenoceptor agonist isoprenaline (0.05-0.15 nmol/kg) and agonists at the high- and low-affinity state of beta1-adrenoceptors, that is, prenalterol (0.3-3 nmol/kg) and (+/-)-4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazole-2-one (CGP 12177; 3-6 nmol/kg), respectively, and at beta2-adrenoceptors, that is, fenoterol (1-5 nmol/kg), increased heart rate by 50 to 60 beats/min. Administration of LPS (0.4, 1, and 1.5 mg/kg), under continuous infusion of vasopressin, dose-dependently amplified the chronotropic response to isoprenaline, prenalterol, and fenoterol (by 80%, 50%, and 100%, respectively) but not to CGP 12177. The beta2-adrenoceptor antagonist erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol (ICI 118551 0.1 mumol/kg) did not affect the chronotropic responses of isoprenaline, fenoterol, and prenalterol under non-endotoxic conditions, but abolished the potentiation of tachycardia produced by LPS (1.5 mg/kg). The beta1-adrenoceptor antagonist (+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]-phenoxy]propyl]-amino]ethoxy]-benzamide CGP 20712A; 0.1 mumol/kg almost completely reduced the chronotropic effects of isoprenaline, fenoterol, and prenalterol both in control rats and in animals exposed to LPS (1.5 mg/kg). We conclude that LPS sensitizes cardiac beta-adrenoceptors by recruiting functionally active beta2-adrenoceptors, but the amplification of tachycardia occurs only when both beta1- and beta2-adrenoceptors are concomitantly activated. The pithed rat may serve as a model to examine the beta-adrenoceptor supersensitivity in vivo.

Ligand-independent negative chronotropic responses of rat and mouse right atria to β-adrenoceptor antagonists

Negative chronotropic effects of beta-adrenoceptor (betaAR) antagonists on right atria from reserpine-treated rats and mice were determined as a test of their inverse agonist activities. BetaAR antagonist ICI-118,551 and nonselective betaAR antagonists alprenolol, propranolol, and timolol produced negative chronotropic effects. In contrast, nonselective betaAR antagonists pindolol and nadolol as well as beta1AR-selective antagonists atenolol, acebutolol, and metoprolol did not cause a significant decrease in atrial rates. The neutral antagonist pindolol but not the inverse agonist alprenolol inhibited the negative chronotropic activities of ICI-118,551. Isoprenaline, salbutamol, and noradrenaline produced positive chronotropic effects; the chronotropic effects of isoprenaline and salbutamol but not of noradrenaline were antagonized by ICI-118,551. It is concluded that both beta1AR and beta2AR mediate positive chronotropic effects of catecholamines on rat and mouse atria but only beta2AR are constitutively active.

Monoamines and the isolated auricle of sepia officinalis: are there &bgr;-like receptors in the heart of a cephalopod?

Pharmacological examinations of isolated auricles from Sepia officinalis were carried out to analyze the putative role of the monoaminergic transmitter/receptor system in the control of auricle function. In conjunction with histofluorescence studies and HPLC analyses, evidence of a double excitatory serotonergic and noradrenergic innervation of the auricles was obtained. Serotonin-induced positive chronotropic and inotropic effects were blocked by mianserin (5-HT1 and 5-HT2) but not by cyproheptadine (5-HT2). It is assumed that the auricular serotonin (5-HT) receptor represents a 5-HT1-like subtype and is not identical to the ventricular 5-HT receptor. Noradrenaline, adrenaline and dopamine evoked mainly positive chronotropic reactions and less prominent positive inotropic reactions. The potency range (pD2 frequency: noradrenaline 6.65 >> adrenaline 5.69 > dopamine 5.34; pD2 amplitude: noradrenaline 6.09 (greater than or equal to) adrenaline 5.91 > dopamine 5.33) indicates out that noradrenaline might be the effective neurotransmitter in vivo. The &agr; -mimetics clonidine ( &agr; 2) and phenylephrine ( &agr; 1) induced positive chronotropic and inotropic effects, while the &bgr;-mimetics albuterol (&bgr;2>&bgr;1) and dobutamine (&bgr;1) revealed only positive inotropic reactions. The &bgr;-agonist isoprenaline mimicked the positive chronotropic effects of noradrenaline and induced the strongest positive inotropic effects of all the agonists tested. Urapidil ( &agr; 1) or phentolamine ( &agr; 1 and &agr; 2) blocked only the positive chronotropic effects of noradrenaline and isoprenaline. The positive inotropic effects of isoprenaline could be blocked by the adenylate cyclase inhibitors MDL-12,330A or SQ-22, 536, which had no effect on the chronotropic effects of isoprenaline. These results suggest that two catecholaminergic receptors are present in the auricles of Sepia officinalis: an &agr; -like adrenoreceptor mediating mainly chronotropic effects, and a &bgr;-like receptor which appears to mediate inotropic effects by activating the cyclic AMP pathway. These results suggest that the auricles exert a regulatory effect on ventricular performance.

Ligand-independent negative chronotropic responses of rat and mouse right atria to β-adrenoceptor antagonists

Negative chronotropic effects of beta-adrenoceptor (betaAR) antagonists on right atria from reserpine-treated rats and mice were determined as a test of their inverse agonist activities. BetaAR antagonist ICI-118,551 and nonselective betaAR antagonists alprenolol, propranolol, and timolol produced negative chronotropic effects. In contrast, nonselective betaAR antagonists pindolol and nadolol as well as beta1AR-selective antagonists atenolol, acebutolol, and metoprolol did not cause a significant decrease in atrial rates. The neutral antagonist pindolol but not the inverse agonist alprenolol inhibited the negative chronotropic activities of ICI-118,551. Isoprenaline, salbutamol, and noradrenaline produced positive chronotropic effects; the chronotropic effects of isoprenaline and salbutamol but not of noradrenaline were antagonized by ICI-118,551. It is concluded that both beta1AR and beta2AR mediate positive chronotropic effects of catecholamines on rat and mouse atria but only beta2AR are constitutively active.

Anti-arrhythmic profile of a garlic dialysate assayed in dogs and isolated atrial preparations

The effects of garlic ( Allium sativum L., Liliaceae) dialysate were studied on arrhythmias induced in anaesthetized dogs and on isolated left rat atria. Garlic dialysate suppressed premature ventricular contractions (PVC) and ventricular tachycardia (VT) in ouabain-intoxicated dogs as well as the ectopic rhythms induced by isoprenaline (10 −6 M) and aconitine (10 −8 M) on electrically driven left rat atria. The effective refractory period (ERP) and the sinus node recovery time (SNRT) of isolated rat atria were prolonged in a dose-dependent manner by the administration of this extract. Garlic dialysate decreased the positive inotropic and chronotropic effects of isoprenaline in a concentration-dependent manner. These last effects were increased by propranolol. The results suggest that garlic dialysate has a significant anti-arrhythmic effect in both ventricular and supraventricular arrhythmias.

Do β<sub>3</sub>-adrenoceptors mediate metabolic responses to isoprenaline

We investigated whether the putative beta 3-adrenoceptors mediated metabolic responses to isoprenaline. Seven normal volunteers received infusions of isoprenaline, a (beta 1, beta 2 and beta 3-agonist), at 0.5-3.0 micrograms/min. They were pretreated with either placebo, 25 mg atenolol (a selective beta 1 antagonist), or 5, 20 and 80 mg nadolol (which blocks beta 1 and beta 2 but not beta 3-adrenoceptors). Isoprenaline markedly (30.6%) increased basal metabolic rate (BMR): this increase was significantly reduced by 25 mg atenolol but not by 5 mg nadolol. Significant beta 2-blockade (from tremor data) occurred with 5 mg nadolol but not with 25 mg atenolol. This suggests that beta 1 but not beta 2-adrenoceptors are involved in the mediating thermogenic effects of isoprenaline. However, the rise in BMR was not totally blocked even by 80 mg nadolol (9.5%), which produced complete beta 1/beta 2 blockade, as evidenced by the elimination of the chronotropic effect of isoprenaline. This implies that the thermogenic response has a non-beta 1/beta 2-mediated component. There were also significant increases in plasma free fatty acids, glycerol, glucose, insulin and lactate, but these were completely abolished by beta 1/beta 2 blockade. Overall, isoprenaline produced an increase in BMR which is only partly due to stimulation of beta 1-adrenoceptors, and which is not associated with beta 1/beta 2-mediated effects on carbohydrate and fat metabolism. This suggests the possibility of thermogenic beta 3-adrenoceptors in man, although their location and role remain unknown.

Non‐uniformity of two‐dimensional myocardial deformation in response to chronotropic and inotropic stimulation in cats

The degree of uniformity of myocardial deformation for cross-oriented segments in the anterior wall of the left ventricle is influenced both by loading conditions and by infusion of isoprenaline. The aim of this study was to clarify the chronotropic influence (atrial pacing) compared to the combined inotropic and chronotropic effects of isoprenaline on uniformity of contraction. In eight open-chest pentobarbitone-anaesthetized cats segment performance was measured by orthogonal sonomicrometry. Heart rate (HR) increased from control state, 199 +/- 5 (mean +/- SEM) beats min-1, to 224 +/- 6 and 227 +/- 7 beats min-1 during atrial pacing and isoprenaline infusion, respectively (P < 0.0005). Circumferential segment shortening remained unchanged during pacing but increased with isoprenaline (P < 0.0005). Longitudinal segments showed reduced shortening during pacing (P < 0.05), whereas shortening during isoprenaline infusion did not differ from control shortening. The ratio between shortening of longitudinal and circumferential segments, long/circ ratio, changed from 0.52 +/- 0.13 in the control state to 0.36 +/- 0.10 during pacing and 0.39 +/- 0.11 with isoprenaline (P < 0.05). End-systolic pressure-length relations were unchanged by atrial pacing but showed leftward shifts during isoprenaline infusion. Myocardial tissue blood flow (microspheres) was unchanged during interventions. We conclude that both atrial pacing and isoprenaline infusion lead to a more non-uniform deformation in the anterior wall. However, non-uniformity during atrial pacing was primarily related to reduced longitudinal shortening whereas the non-uniformity during isoprenaline infusion was due to increased circumferential shortening.

The effect of vanadate on alloxan-diabetic rat atria

In this study, we examined the effect of vanadate treatment on cardiac changes recognized in diabetic rats. In addition, the possible contribution of thyroid hormones to vanadate's effect on alloxan-diabetic atria was also investigated. Administration of alloxan to rats, as expected, resulted in hyperglycemia; hypoinsulinemia reduced thyroid hormone levels, decreased body weight and depressed cardiac function. Vanadate treatment of diabetic rats normalized blood glucose and serum thyroid hormone levels, neither were serum insulin levels of diabetic animals corrected after vanadate treatment. Vanadate treatment, however, did not affect the body weights of diabetic rats. Spontaneously-beating atria from diabetic rats were found to have decreased rates but increased forces of contractions compared with those from controls. On the other hand, the responsiveness of diabetic atria to both inotropic and chronotropic effects of isoprenaline was found to be decreased. Vanadate treatment resulted in the normalization of these alterations observed in diabetic atria. These results thus indicate that the normalizing effect of vanadate on diabetes-induced hypothyroidism may contribute to its effect in preventing cardiac changes observed at the early stage of diabetes.

The α‐ and β‐adrenoceptor blocking activities of labetalol and its RR‐SR (50:50) stereoisomers

1. We compared the alpha 1-, alpha 2- and beta 1-adrenoceptor blocking potencies of labetalol with those of its two stereoisomers (RR and SR) in pithed rats and in homogenized rat cerebral cortex and heart. 2. In pithed rats, labetalol and the RR-SR combination were given orally either at doses of 25 and 50 mg kg-1 body wt. or intravenously at doses of 1 and 5 mg kg-1 body wt. Prazosin 4 and 20 micrograms kg-1 body wt. and propranolol 1 and 5 mg kg-1 body wt., were given intravenously for comparison studies of potency at alpha 1- and beta 1-adrenoceptors, respectively. Effects were studied before and after i.v. administration of either phenylephrine (at doses which increased the mean arterial pressure by approximately 80 mmHg) or isoprenaline (at doses that increased heart rate by approximately 100 beats min-1). 3. In pithed rats, labetalol and the RR-SR combination antagonized, in a dose-dependent manner, the pressor effect of phenylephrine (P less than 0.05) and the chronotropic effect of isoprenaline (P less than 0.05). Following both oral and intravenous dosing, the RR-SR combination was twice potent as labetalol in terms of alpha 1- and beta 1-adrenoceptor antagonism at equivalent doses. 4. Labetalol and the enantiomers lacked affinity at alpha 2-adrenoceptors while at alpha 1-adrenoceptors the order of potency was prazosin much greater than RR-SR greater than labetalol. At beta 1-adrenoceptors, the affinity of the compound RR-SR was about 3 times that of labetalol.5. As labetalol is a mixture of active (RR and SR) and inactive (SS and SR) enantiomers (in terms of alpha and beta receptor actions), the combination of RR and SR may be a valuable substitute for labetalol in the treatment of systemic hypertension. Although the potential for non-specific side effects (common to all four enantiomers) could be expected to be diminished, recent reports by postmarketing surveillance indicate that the RR isomer (dilevalol) can induce liver toxicity. Interestingly, labetalol is devoid of this effect; whether the combination of RR and SR enantiomers could be of clinical importance warrants further investigation.

Thyroid hormones mediated effect of insulin on alloxan diabetic rat atria

1. The influence of alloxan-induced diabetes was studied on spontaneously beating rat atria. Diabetic atria were found to have decreased rates, increased contractility and decreased responsiveness to both inotropic and chronotropic effects of isoprenaline. 2. Thyroid hormone levels were significantly reduced in diabetic animals. This revealed that the decrease in atrial β-adrenergic responses was associated with a reduction in serum levels of thyroid hormones. 3. Insulin treatment of diabetic rats for 10 days corrected the changes observed in diabetic atria. Serum levels of thyroid hormones returned to normal following insulin treatment as well. 4. Administration of insulin to thyroidectomized-diabetic rats did not reverse the diabetes-induced changes suggesting that thyroid hormones are needed for insulin to normalize the alterations observed in diabetic atria.

Actions of alinidine and AQ-A 39 on rate and contractility of guinea pig atria during beta-adrenoceptor stimulation.

We studied two new agents, alinidine (St 567, 2-[N-allyl-N-(2,6-dichlorophenyl)-amino]-2-imidazoline) and AQ-A 39 (5,6-dimethoxy-2-[3[[alpha-(3,4-dimethoxy)-phenylethyl] methylamino]propyl]phthalimidine), in isolated guinea pig atria with respect to their specificity to decrease heart rate but not contractility during beta-adrenoceptor stimulation. Spontaneous electrical activity in sinoatrial node preparations was increased by perfusion with isoprenaline (0.1 micrograms/ml); addition of alinidine (3 micrograms/ml), AQ-A 39 (3 micrograms/ml), or propranolol (0.03-0.3 micrograms/ml) reduced sinus rate to the control values. The same concentrations of these drugs were tested in electrically driven (1 Hz) left atria. The positive inotropic effect of isoprenaline was not affected by alinidine and AQ-A 39; however, it was markedly reduced or abolished by propranolol. The experiments, therefore, demonstrated the bradycardic specificity of alinidine and AQ-A 39 under conditions of beta-adrenoceptor stimulation. A cumulative concentration-response curve for the positive chronotropic effect of isoprenaline was established in spontaneously beating atria. AQ-A 39, 1 and 10 micrograms/ml, reduced the control sinus rate but did not affect the following concentration-response curve of isoprenaline. A similar result was published earlier for alinidine and excludes an interaction of both drugs with isoprenaline on a possible subgroup of selective beta-adrenoceptors in the sinoatrial node.

A Comparative Study of the Effects of OPC-1085 and Propranolol on Isolated Guinea Pig Atrium

Effects of a newly synthesized beta-blocker, 5-(3-tert-butylamino-2-hydroxy) propoxy-3, 4-dihydrocarbostyril (OPC-1085) were compared with those of propranolol. OPC-1085 had a potency about 3 times greater than that of propranolol in blocking the positive inotropic and chronotropic effects of isoprenaline on the isolated guinea pig atrium. At a concentration of over 3×10−5 M, OPC-1085 produced negative inotropic and chronotropic effects. However, these effects were about 10 times weaker than those of propranolol. Suppressing effects on the rate of rise and on the maximum driving frequency of action potentials were also more than 10 times less than those of propranolol. There was almost no change in the action potential of vagus nerve after a 10 min treatment with OPC-1085 (10−5 M), while the action potential was reduced to 60–70% with propranolol (10−5 M).

Pharmacokinetics and pharmacodynamics of alprenolol in the treatment of hypertension. I. Relationship between plasma concentration and adrenergic beta-receptor blockade.

Mean steady-state plasma concentrations of alprenolol were studied in relationship to the degree of beta-blockade, in sixteen patients receiving 600 mg daily in divided doses. Steady-state alprenolol concentrations were determined from the area under the plasma concentration-time curve during one eight-hour dosage interval after treatment for six weeks. Beta-blockade during alprenolol treatment was assessed from the chronotropic response to intravenous isoprenaline compared to the response after six weeks of placebo therapy. Although there was interindividual variability in the mean steady-state alprenolol concentration (range 11 - 141 ng/ml), and in the degree of beta-blockade (7-fold), the correlation between the two variables was highly significant (r = 0.80, p less than 0.001). The prescribed dose of alprenolol (mg/kg) was not significantly correlated with the plasma level of alprenolol or the beta-blockade. The chronotropic effects of isoprenaline during placebo and alprenolol were significantly interrelated (r = 0.79, p less than 0.001).

Influence of INPEA, pindolol and propranolol on the chronotropic and metabolic responses to β-adrenergic stimulation in intact rats

A study was made of the effect of isoprenaline given intraperitoneally to rats on the chronotropic effect and the activation of myocardial phosphorylase. Pretreatment with three β-blocking agents: pindolol, propranolol and INPEA was carried out to separate metabolic and chronotropic effects of isoprenaline on heart. Isoprenaline at 0.01–40 mg/kg augmented heart rate, but the activation of phosphorylase started only from the dose of 0.02 mg/kg. Metabolic effect of isoprenaline reached the maximum at a lower dose (0.1 mg/kg) than chronotropic effect of isoprenaline did (1 mg/kg). Pindolol at 0.03 mg/kg, propranolol in doses 0.5–5 mg/kg and INPEA at a dose of 50 mg/kg slowed heart rate but did not influence phosphorylase activity. INPEA in doses 0.5–5 mg/kg, accelerated heart rate, but did not activate myocardial phosphorylase. β-Blocking potency of all three drugs was nearly the same with respect to metabolic and chronotropic response to isoprenaline, but only when measured at the level of 50 per cent of inhibition. 1050 were: pindolol —0.006 mg/kg, propranolol —0.33 mg/kg and INPEA —2.8 mg/kg, however distinct differences between metabolic and chronotropic blockade in other doses of propranolol and INPEA were found. Linear dose dependent β-adrenergic blockade was induced with respect to metabolic and chronotropic effects by pindolol, with respect to chronotropic effect by propranolol and in respect to metabolic effect by INPEA. The blockade of metabolic effect by propranolol and the blockade of chronotropic effect by INPEA occurred abruptly from the zero level to maximal level when the dosage of these drugs was doubled.