Using Esterase Selectivity to Determine the In Vivo Duration of Systemic Availability and Abolish Systemic Side Effects of Topical ß-Blockers.

For disorders of the skin, eyes, ears, and respiratory tract, topical drugs, delivered directly to the target organ, are a therapeutic option. Compared with systemic oral therapy, the benefits of topical treatments include a faster onset of action, circumventing the liver first pass drug metabolism, and reducing systemic side effects. Nevertheless, some systemic absorption still occurs for many topical agents resulting in systemic side effects. One way to prevent these would be to develop drugs that are instantly degraded upon entry into the bloodstream by serum esterases. Because topical ß-blockers are used in glaucoma and infantile hemeangioma and cause systemic side effects, the ß-adrenoceptor system was used to test this hypothesis. Purified liver esterase reduced the apparent affinity of esmolol, an ester-containing ß-blocker used in clinical emergencies, for the human ß-adrenoceptors in a concentration and time-dependent manner. However, purified serum esterase had no effect on esmolol. Novel ester-containing ß-blockers were synthesized and several were sensitive to both liver and serum esterases. Despite good in vitro affinity, one such compound, methyl 2-(3-chloro-4-(3-((2-(3-(3-chlorophenyl)ureido)ethyl)amino)-2-hydroxypropoxy)phenyl)acetate, had no effect on heart rate when injected intravenously into rats, even at 10 times the equipotent dose of esmolol and betaxolol that caused short and sustained reductions in heart rate, respectively. Thus, ester-based drugs, sensitive to serum esterases, offer a mechanism for developing topical agents that are truly devoid of systemic side effects. Furthermore, differential susceptibility to liver and serum esterases degradation may also allow the duration of systemic availability for other drugs to be fine-tuned.

Peroxisome proliferator-activated receptor γ agonism attenuates endotoxaemia-induced muscle protein loss and lactate accumulation in rats.

The peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (Rosi) appears to provide protection against organ dysfunction during endotoxaemia. We examined the potential benefits of Rosi on skeletal muscle protein maintenance and carbohydrate metabolism during lipopolysaccharide (LPS)-induced endotoxaemia. Sprague-Dawley rats were fed either standard chow (control) or standard chow containing Rosi (8.5 ± 0.1 mg·kg-1·day-1) for 2 weeks before and during 24 h continuous intravenous infusion of LPS (15 µg·kg-1·h-1) or saline. Rosi blunted LPS-induced increases in muscle tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA by 70% (P<0.05) and 64% (P<0.01) respectively. Furthermore, Rosi suppressed the LPS-induced reduction in phosphorylated AKT and phosphorylated Forkhead box O (FOXO) 1 protein, as well as the up-regulation of muscle RING finger 1 (MuRF1; P<0.01) mRNA and the LPS-induced increase in 20S proteasome activity (P<0.05). Accordingly, LPS reduced the muscle protein:DNA ratio (∼30%, P<0.001), which Rosi offset. Increased muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA (P<0.001) and muscle lactate accumulation (P<0.001) during endotoxaemia were suppressed by Rosi. Thus, pre-treatment with Rosi reduced muscle cytokine accumulation and blunted muscle protein loss and lactate accumulation during endotoxaemia, and at least in part by reducing activation of molecular events known to increase muscle protein breakdown and mitochondrial pyruvate use.

Novel selective ß1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease.

ß-Blockers reduce mortality and improve symptoms in people with heart disease; however, current clinically available ß-blockers have poor selectivity for the cardiac ß1-adrenoceptor (AR) over the lung ß2-AR. Unwanted ß2-blockade risks causing life-threatening bronchospasm and reduced efficacy of ß2-agonist emergency rescue therapy. Thus, current life-prolonging ß-blockers are contraindicated in patients with both heart disease and asthma. Here, we describe NDD-713 and -825, novel highly ß1-selective neutral antagonists with good pharmaceutical properties that can potentially overcome this limitation. Radioligand binding studies and functional assays that use human receptors expressed in Chinese hamster ovary cells demonstrate that NDD-713 and -825 have nanomolar ß1-AR affinity >500-fold ß1-AR vs ß2-AR selectivity and no agonism. Studies in conscious rats demonstrate that these antagonists are orally bioavailable and cause pronounced ß1-mediated reduction of heart rate while showing no effect on ß2-mediated hindquarters vasodilatation. These compounds also have good disposition properties and show no adverse toxicologic effects. They potentially offer a truly cardioselective ß-blocker therapy for the large number of patients with heart and respiratory or peripheral vascular comorbidities.-Baker, J. G., Gardiner, S. M., Woolard, J., Fromont, C., Jadhav, G. P., Mistry, S. N., Thompson, K. S. J., Kellam, B., Hill, S. J., Fischer, P. M. Novel selective ß1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease.

Excess maternal salt or fructose intake programmes sex-specific, stress- and fructose-sensitive hypertension in the offspring.

The Western diet is typically high in salt and fructose, which have pressor activity. Maternal diet can affect offspring blood pressure, but the extent to which maternal intake of excess salt and fructose may influence cardiovascular function of the offspring is unknown. We sought to determine the effect of moderate maternal dietary intake of salt and/or fructose on resting and stimulated cardiovascular function of the adult male and female offspring. Pregnant rats were fed purified diets (± 4% salt) and water (± 10% fructose) before and during gestation and through lactation. Male and female offspring were weaned onto standard laboratory chow. From 9 to 14 weeks of age, cardiovascular parameters (basal, circadian and stimulated) were assessed continuously by radiotelemetry. Maternal salt intake rendered opposite-sex siblings with a 25-mmHg difference in blood pressure as adults; male offspring were hypertensive (15 mmHg mean arterial pressure (MAP)) and female offspring were hypotensive (10 mmHg MAP) above and below controls, respectively. Sex differences were unrelated to endothelial nitric oxide activity in vivo, but isolation-induced anxiety revealed a significantly steeper coupling between blood pressure and heart rate in salt-exposed male offspring but not in female offspring. MAP of all offspring was refractory to salt loading but sensitive to subsequent dietary fructose, an effect exacerbated in female offspring from fructose-fed dams. Circadian analyses of pressure in all offspring revealed higher mean set-point for heart rate and relative non-dipping of nocturnal pressure. In conclusion, increased salt and fructose in the maternal diet has lasting effects on offspring cardiovascular function that is sex-dependent and related to the offspring's stress-response axis.

Excess maternal salt intake produces sex-specific hypertension in offspring: putative roles for kidney and gastrointestinal sodium handling.

Hypertension is common and contributes, via cardiovascular disease, towards a large proportion of adult deaths in the Western World. High salt intake leads to high blood pressure, even when occurring prior to birth - a mechanism purported to reside in altered kidney development and later function. Using a combination of in vitro and in vivo approaches we tested whether increased maternal salt intake influences fetal kidney development to render the adult individual more susceptible to salt retention and hypertension. We found that salt-loaded pregnant rat dams were hypernatraemic at day 20 gestation (147±5 vs. 128±5 mmoles/L). Increased extracellular salt impeded murine kidney development in vitro, but had little effect in vivo. Kidneys of the adult offspring had few structural or functional abnormalities, but male and female offspring were hypernatraemic (166±4 vs. 149±2 mmoles/L), with a marked increase in plasma corticosterone (e.g. male offspring; 11.9 [9.3-14.8] vs. 2.8 [2.0-8.3] nmol/L median [IQR]). Furthermore, adult male, but not female, offspring had higher mean arterial blood pressure (effect size, +16 [9-21] mm Hg; mean [95% C.I.]. With no clear indication that the kidneys of salt-exposed offspring retained more sodium per se, we conducted a preliminary investigation of their gastrointestinal electrolyte handling and found increased expression of proximal colon solute carrier family 9 (sodium/hydrogen exchanger), member 3 (SLC9A3) together with altered faecal characteristics and electrolyte handling, relative to control offspring. On the basis of these data we suggest that excess salt exposure, via maternal diet, at a vulnerable period of brain and gut development in the rat neonate lays the foundation for sustained increases in blood pressure later in life. Hence, our evidence further supports the argument that excess dietary salt should be avoided per se, particularly in the range of foods consumed by physiologically immature young.

Maternal fructose and/or salt intake and reproductive outcome in the rat: effects on growth, fertility, sex ratio, and birth order.

Maternal diet can significantly skew the secondary sex ratio away from the expected value of 0.5 (proportion males), but the details of how diet may do this are unclear. Here, we altered dietary levels of salt (4% salt in the feed) and/or fructose (10% in the drinking water) of pregnant rats to model potential effects that consumption of a "Western diet" might have on maternofetal growth, development, and sex ratio. We demonstrate that excess fructose consumption before and during pregnancy lead to a marked skew in the secondary sex ratio (proportion of males, 0.60; P < 0.006). The effect was not mediated by selective developmental arrest of female embryos or influenced by fetal position in the uterine horn or sex-specific effects on sperm motility, suggesting a direct effect of glycolyzable monosaccharide on the maternal ovary and/or ovulated oocyte. Furthermore, combined excess maternal consumption of salt and fructose-sweetened beverage significantly reduced fertility, reflected as a 50% reduction in preimplantation and term litter size. In addition, we also noted birth order effects in the rat, with sequential implantation sites tending to be occupied by the same sex.

Synthesis and in vitro and in vivo characterization of highly ß1-selective ß-adrenoceptor partial agonists.

ß-Adrenoceptor antagonists boast a 50-year use for symptomatic control in numerous cardiovascular diseases. One might expect highly selective antagonists are available for the human ß-adrenoceptor subtype involved in these diseases, yet few truly ß1-selective molecules exist. To address this clinical need, we re-evaluated LK 204-545 (1), (1) a selective ß1-adrenoceptor antagonist, and discovered it possessed significant partial agonism. Removal of 1's aromatic nitrile afforded 19, a ligand with similar ß1-adrenoceptor selectivity and partial agonism (log KD of -7.75 and -5.15 as an antagonist of functional ß1- and ß2-mediated responses, respectively, and 34% of the maximal response of isoprenaline (ß1)). In vitro ß-adrenoceptor selectivity and partial agonism of 19 were mirrored in vivo. We designed analogues of 19 to improve affinity, selectivity, and partial agonism. Although partial agonism could not be fully attenuated, SAR suggests that an extended alkoxyalkoxy side chain, alongside substituents at the meta- or para-positions of the phenylurea, increases ligand affinity and ß1-selectivity.

Predicting in vivo cardiovascular properties of ß-blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses.

ß-Adrenoceptor antagonists differ in their degree of partial agonism. In vitro assays have provided information on ligand affinity, selectivity, and intrinsic efficacy. However, the extent to which these properties are manifest in vivo is less clear. Conscious freely moving rats, instrumented for measurement of heart rate (ß1; HR) and hindquarters vascular conductance (ß2; HVC) were used to measure receptor selectivity and ligand efficacy in vivo. CGP 20712A caused a dose-dependent decrease in basal HR (P<0.05, ANOVA) at 5 doses between 6.7 and 670 µg/kg (i.v.) and shifted the dose-response curve for isoprenaline to higher agonist concentrations without altering HVC responses. In contrast, at doses of 67 µg/kg (i.v.) and above, ICI 118551 substantially reduced the HVC response to isoprenaline without affecting HR responses. ZD 7114, xamoterol, and bucindolol significantly increased basal HR (ΔHR: +122 ± 12, + 129 ± 11, and + 59 ± 11 beats/min, respectively; n=6), whereas other ß-blockers caused significant reductions (all at 2 mg/kg i.v.). The agonist effects of xamoterol and ZD 7114 were equivalent to that of the highest dose of isoprenaline. Bucindolol, however, significantly antagonized the response to the highest doses isoprenaline. An excellent correlation was obtained between in vivo and in vitro measures of ß1-adrenoceptor efficacy (R(2)=0.93; P<0.0001).

Low-dose dexamethasone prevents endotoxaemia-induced muscle protein loss and impairment of carbohydrate oxidation in rat skeletal muscle.

We recently provided evidence suggesting a role for cytokine-mediated inhibition of Akt/Forkhead box O 1 (FOXO1) signalling in the induction of muscle atrophy and impairment of muscle carbohydrate oxidation during lipopolysaccharide (LPS)-induced endotoxaemia in rats. We hypothesized that a low-dose dexamethasone (Dex; anti-inflammatory agent) infusion during endotoxaemia would prevent the LPS-induced impairment of Akt/FOXO1 signalling, and therefore prevent the muscle atrophy and impairment of carbohydrate oxidation. Chronically instrumented Sprague-Dawley rats received a continuous intravenous infusion of LPS (15 microg kg(-1) h(-1)), Dex (12.5 microg kg(-1) h(-1)), Dex+LPS or saline for 24 h at 0.4 ml h(-1). LPS infusion caused haemodynamic changes consistent with a hyperdynamic circulation and induced increases in muscle tumour necrosis factor-alpha (TNF-alpha; 10-fold, P < 0.001), interleukin-6 (IL-6; 14-fold, P < 0.001) and metallothionein-1A (MT-1A; 187-fold, P < 0.001) mRNA expression. Dex co-administration abolished most of the haemodynamic effects of LPS and reduced the increase in muscle TNF-alpha, IL-6 and MT-1A by 51% (P < 0.01), 85% (P < 0.001) and 58% (P < 0.01), respectively. Dex infusion during endotoxaemia also prevented the LPS-induced 40% reduction in the muscle protein:DNA ratio and decrease in Akt phosphorylation, and partially prevented the reduction in FOXO1 phosphorylation. However, Dex did not prevent the LPS-mediated increase in muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) mRNA expression, but did significantly reduce the LPS-mediated increase in cathepsin-L mRNA expression and enzyme activity by 43% (P < 0.001) and 53% (P < 0.05), respectively. Furthermore, Dex suppressed LPS-induced pyruvate dehydrogenase kinase 4 (PDK4) mRNA upregulation by approximately 50% (P < 0.01), and prevented LPS-mediated muscle glycogen breakdown and lactate accumulation. Thus, low-dose Dex infusion during endotoxaemia prevented muscle atrophy and the impairment of carbohydrate oxidation, potentially through suppression of cytokine-mediated Akt/FOXO inhibition, and blunting of cathepsin-L-mediated lysosomal protein breakdown.

Effects of endotoxaemia on protein metabolism in rat fast-twitch skeletal muscle and myocardium.

BACKGROUND: It is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically observed in fast-twitch skeletal muscle during endotoxaemia. METHODOLOGY/PRINCIPAL FINDINGS: To investigate this further, and to determine if there is any divergence in the response of skeletal muscle and myocardium in the mechanisms that are thought to be largely responsible for eliciting changes in protein content, Sprague Dawley rats were implanted with vascular catheters and administered lipopolysaccharide (LPS; 150 microg kg(-1) h(-1)) intravenously for 2 h, 6 h or 24 h (saline administered control animals were also included), after which the extensor digitorum longus (EDL) and myocardium were removed under terminal anaesthesia. The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P<0.05), but was no different from controls in the myocardium. At the same time point, a significant increase in MAFbx/atrogin-1 and MuRF1 mRNA (3.7+/-0.7- and 19.5+/-1.9-fold increase vs. controls, respectively; P<0.05), in addition to protein levels of alpha1-3, 5-7 subunits of the 20S proteasome, were observed in EDL but not myocardium. In contrast, elevations in phosphorylation of p70 S6K residues Thr(421)/Ser(424), and 4E-BP1 residues Thr(37)/Thr(46) (P<0.05), consistent with an elevation in translation initiation, were seen exclusively in the myocardium of LPS-treated animals. CONCLUSIONS/SIGNIFICANCE: In summary, these findings suggest that the myocardium does not undergo the same catabolic response as skeletal muscle during early endotoxaemia, partly due to the absence of transcriptional and signalling events in the myocardium typically associated with increased muscle proteolysis and the suppression of protein synthesis.

Acute hypertension reveals depressor and vasodilator effects of cannabinoids in conscious rats.

BACKGROUND AND PURPOSE: The cardiovascular effects of cannabinoids can be influenced by anaesthesia and can differ in chronic hypertension, but the extent to which they are influenced by acute hypertension in conscious animals has not been determined. EXPERIMENTAL APPROACH: We examined cardiovascular responses to intravenous administration of anandamide and the synthetic cannabinoid, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), in conscious male Wistar rats made acutely hypertensive by infusion of angiotensin II (AII) and arginine vasopressin (AVP). Rats were chronically instrumented for measurement of arterial blood pressure and vascular conductances in the renal, mesenteric and hindquarters beds. KEY RESULTS: Anandamide dose-dependently decreased the mean arterial blood pressure of rats made hypertensive by AII-AVP infusion, but not normotensive rats. Interestingly, acute hypertension also revealed a hypotensive response to WIN55212-2, which caused hypertension in normotensive animals. The enhanced depressor effects of the cannabinoids in acute hypertension were associated with increased vasodilatation in hindquarters, renal and mesenteric vascular beds. Treatment with URB597, which inhibits anandamide degradation by fatty acid amide hydrolase, potentiated the depressor and mesenteric vasodilator responses to anandamide. Furthermore, haemodynamic responses to WIN55212-2, but not to anandamide, were attenuated by the CB(1) receptor antagonist, AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide]. CONCLUSIONS AND IMPLICATIONS: These results broadly support the literature showing that the cardiovascular effects of cannabinoids can be exaggerated in hypertension, but highlight the involvement of non-CB(1) receptor-mediated mechanisms in the actions of anandamide.

A potential role for Akt/FOXO signalling in both protein loss and the impairment of muscle carbohydrate oxidation during sepsis in rodent skeletal muscle.

Sepsis causes muscle atrophy and insulin resistance, but the underlying mechanisms are unclear. Therefore, the present study examined the effects of lipopolysaccharide (LPS)-induced endotoxaemia on the expression of Akt, Forkhead Box O (FOXO) and its downstream targets, to identify any associations between changes in FOXO-dependent processes influencing muscle atrophy and insulin resistance during sepsis. Chronically instrumented male Sprague-Dawley rats received a continuous intravenous infusion of LPS (15 microg kg(-1) h(-1)) or saline for 24 h at 0.4 ml h(-1). Animals were terminally anaesthetized and the extensor digitorum longus muscles from both hindlimbs were removed and snap-frozen. Measurements were made of mRNA and protein expression of selected signalling molecules associated with pathways regulating protein synthesis and degradation and carbohydrate metabolism. LPS infusion induced increases in muscle tumour necrosis factor-alpha (8.9-fold, P < 0.001) and interleukin-6 (8.4-fold, P < 0.01), paralleled by reduced insulin receptor substrate-1 mRNA expression (-0.7-fold, P < 0.01), and decreased Akt1 protein and cytosolic FOXO1 and FOXO3 phosphorylation. These changes were accompanied by significant increases in muscle atrophy F-box mRNA (5.5-fold, P < 0.001) and protein (2-fold, P < 0.05) expression, and pyruvate dehydrogenase kinase 4 mRNA (15-fold, P < 0.001) and protein (1.6-fold, P < 0.05) expression. There was a 29% reduction in the muscle protein: DNA ratio, a 56% reduction in pyruvate dehydrogenase complex (PDC) activity (P < 0.05), and increased glycogen degradation and lactate accumulation. The findings of this study suggest a potential role for Akt/FOXO in the simultaneous impairment of carbohydrate oxidation, at the level of PDC, and up-regulation of muscle protein degradation, in LPS-induced endotoxaemia.

The in vitro and in vivo cardiovascular effects of Delta9-tetrahydrocannabinol in rats made hypertensive by chronic inhibition of nitric-oxide synthase.

Evidence suggests that Delta9-tetrahydrocannabinol (THC) may have antihypertensive effects and that the vasodilator effect of endocannabinoids is enhanced in rats made hypertensive by chronic NO synthase inhibition. Therefore, the aims of the present study were to investigate whether the in vitro and in vivo cardiovascular responses to THC are altered by Nomega-nitro-L-arginine methyl ester (L-NAME) treatment. The vasorelaxant effects of THC were enhanced in small mesenteric arteries from L-NAME-treated rats. This enhanced response was not inhibited by cannabinoid CB1 receptor antagonism [1 microM N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM251]. Pretreating vessels with the transient receptor potential vanilloid receptor receptor agonist capsaicin at 10 microM for 1 h reduced vasorelaxation to THC to a greater extent in L-NAME-treated than control rats. Inhibition of cyclooxygenase with 10 microM indomethacin inhibited THC responses in arteries from L-NAME-treated rats but not from control rats. In conscious, chronically instrumented rats, 1 mg kg-1 i.v. THC caused a pressor effect, with vasoconstriction of the renal and mesenteric vascular beds, and hindquarters vasodilatation. Pretreatment with 3 mg kg-1 i.v. AM251 reduced the pressor and vasoconstrictor effects of THC, abolished the hindquarters vasodilatation, and revealed a bradycardic response. L-NAME-treated rats showed similar pressor and vasoconstrictor responses to THC, but with bradycardia, and reduced hindquarter vasodilatation. These data show that, in vitro, isolated arteries of L-NAME-treated rats show enhanced vasorelaxant responses to THC through an increased sensory nerve component and stimulation of prostanoids. However, in vivo, THC causes a CB1 receptor-mediated pressor effect with hindquarters vasodilatation. There was no evidence of enhanced vasodilator effects of THC in L-NAME-treated animals in vivo.

Mesenteric vasoconstriction and hindquarters vasodilatation accompany the pressor actions of exendin-4 in conscious rats.

The hemodynamic effects of the glucagon-like peptide-1 (GLP-1) receptor agonist, exendin-4, and putative underlying mechanisms were assessed in conscious male Sprague-Dawley rats. At a dose of 25 ng kg(-1) i.v., exendin-4 had little effect, but doses of 250 and 2500 ng kg(-1) had significant tachycardic effects (+66 +/- 9 and +95 +/- 16 beats min(-1) at 5 min, respectively) and pressor actions (+10 +/- 2 and +12 +/- 1 mm Hg), accompanied by substantial falls in mesenteric vascular conductance (-38 +/- 3% and -47 +/- 3%) and increases in hindquarters vascular conductance (+82 +/- 14% and +126 +/- 15%). The latter were likely due to adrenaline-mediated activation of beta(2) adrenoceptors since they were abolished by the beta(2) adrenoceptor antagonist, ICI 118551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol) hydrochloride], or propranolol [(RS)-1-[(1-methylethyl)amino]-3-(1-naphthalenyloxy)-2-propanol], and absent in adrenal-demedullated rats. In the presence of beta-adrenoceptor antagonism, the tachycardic effects of exendin-4 were suppressed, but the pressor action was enhanced. Enhancement of the pressor action of exendin-4 was not seen in adrenal-demedullated rats or in animals given phentolamine in addition to propranolol, consistent with a component of the pressor action of exendin-4 being due to an adrenaline-mediated positive inotropic effect mediated by alpha-adrenoceptors. The mesenteric vasoconstrictor effect of exendin-4 was unaffected by antagonism of alpha-adrenoceptors, vasopressin receptors, angiotensin receptors, or GLP-1 receptors, although antagonism of the latter substantially inhibited the hindquarter vasodilator effects of exendin-4. These results are consistent with exendin-4 having cardiovascular effects through GLP-1 receptor-dependent and -independent mechanisms, some of which involve sympathoadrenal activation.

Regional heterogeneity in the haemodynamic responses to urotensin II infusion in relation to UT receptor localisation.

The aim of the study was to measure regional haemodynamic responses to 6 h infusions of human urotensin II (hUII), to identify possible mediators of the effects observed, and to relate the findings to the distribution of urotensin II receptors (UT receptors). Male, Sprague-Dawley rats had pulsed Doppler flow probes and intravascular catheters implanted for measurement of regional haemodynamics in the conscious, freely moving state. Infusions of saline (0.4 ml h(-1)) or hUII (30, 300 and 3,000 pmol kg(-1) h(-1)) were given i.v. for 6 h, and the effects of pretreatment with indomethacin (5 mg kg(-1) h(-1)), N(G)-nitro-L-arginine methyl ester (L-NAME, 3 mg kg(-1) h(-1)) or propranolol (1 mg kg(-1); 0.5 mg kg(-1) h(-1)) on responses to hUII (300 pmol kg(-1) h(-1) for 6 h) were assessed. Cellular localisation of UT receptor-like immunoreactivity was determined in relevant tissues. hUII caused dose-dependent tachycardia and hindquarters vasodilatation, accompanied by a slowly developing rise in blood pressure. Haemodynamic effects of hUII were attenuated by propranolol or L-NAME and abolished by indomethacin. UT receptor-like immunoreactivity was detected in skeletal and vascular smooth muscle. The findings indicate that in conscious rats, infusions of hUII cause vasodilatation, which, of the vascular beds monitored, is selective for the hindquarters and dependent on cyclooxygenase products and nitric oxide. The pressor effect of hUII under these conditions is likely to be due to an increase in cardiac output, possibly due to a positive inotropic effect. UT receptor-like immunoreactivity present in skeletal muscle is consistent with the haemodynamic pattern.

Regional hemodynamic actions of selective corticotropin-releasing factor type 2 receptor ligands in conscious rats.

In conscious male Sprague-Dawley rats, we compared regional hemodynamic actions of the selective corticotropin-releasing factor type 2 (CRF(2)) receptor ligands human and mouse urocortin 2 (hUCN2 and mUCN2, respectively) with those of CRF. Bolus i.v. doses of 3 and 30 pmol kg(-1) hUCN2, mUCN2, or CRF had no significant hemodynamic actions, but at doses of 300 and 3000 pmol kg(-1), all three peptides caused dose-dependent tachycardia and hypotension, with rapid-onset, short-duration, mesenteric vasodilatation and slower-onset, more prolonged hindquarters vasodilatation but little or no change in renal vascular conductance. Pretreatment with the nonselective CRF receptor antagonist astressin or the selective CRF(2) receptor antagonist antisauvagine 30 abolished all the cardiovascular actions of all three peptides. Indomethacin had no effect on responses to hUCN2, and there was no evidence for any involvement of nitric oxide (NO) in the vasodilator actions of hUCN2. There was no evidence that recruitment of angiotensin- and endothelin-mediated vasoconstrictor mechanisms counteracted the vascular actions of hUCN2. The results indicate that the hemodynamic effects of i.v. hUCN2, mUCN2, and CRF depend on activation of CRF(2) receptors and do not involve NO or prostanoids.

Bolus injection of human UII in conscious rats evokes a biphasic haemodynamic response.

A biphasic cardiovascular response to bolus i.v. injection of human urotensin II (hUII, 3 nmol kg(-1)) in conscious, male, Sprague-Dawley (SD) rats was identified and underlying mechanisms were explored. Initially (0-5 min) there was tachycardia, hypotension and mesenteric and hindquarters vasodilatation; later (30-120 min), tachycardia, hindquarters vasodilatation and a modest rise in blood pressure occurred. Pretreatment with indomethacin or N(G) nitro-l-arginine methylester (l-NAME) reduced the mesenteric vasodilator response to hUII, and abolished the late tachycardia and hindquarters vasodilatation. Indomethacin also abolished the hypotension and early hindquarters vasodilatation, and substantially reduced the initial tachycardia. Indomethacin and l-NAME together prevented all haemodynamic responses to hUII. Inhibition of inducible NOS had no effect on responses to hUII, whereas inhibition of neuronal NOS reduced the delayed tachycardic response to hUII but did not significantly affect the vasodilatation. Only the initial tachycardic response to hUII was antagonised by propranolol. In spontaneously hypertensive rats (SHR), the initial haemodynamic responses to hUII were qualitatively similar to those in SD rats, although there was also a modest renal vasodilatation. The secondary response comprised a smaller tachycardia and a small rise in blood pressure, with no significant hindquarters vasodilatation. Haemodynamic responses to hUII were not enhanced by endothelin and angiotensin receptor antagonism in either SD rats or in SHRs. One interpretation of these results is that the primary response to bolus injection of hUII is prostanoid- or prostanoid- and NO-mediated (mesenteric vasodilatation) and that this triggers secondary events, which are dependent on eNOS (hindquarters vasodilatation) and neuronal NOS (tachycardia).

Regional hemodynamic effects of the N-(2-benzoylphenyl)-L-tyrosine peroxisome proliferator-activated receptor-gamma ligand, GI 262570 [(S)-2-(2-benzoylphenylamino)-3-[4-[2-(5-methyl-2-phenyl-2-oxazol-4-yl)ethoxy]phenyl]propionic acid], in conscious rats.

This study provides novel data on the regional hemodynamic effects of the peroxisome proliferator-activated receptor-gamma activator, GI 262570 [(S)-2-(2-benzoylphenylamino)-3-[4-[2-(5-methyl-2-phenyl-2-oxazol-4-yl)ethoxy]phenyl]propionic acid], in conscious, male Sprague-Dawley rats. Administration of GI 262570 twice daily for 4 days caused a slowly developing, modest fall in mean arterial blood pressure, associated with a progressive, hyperemic hindquarters vasodilatation, but with no consistent changes in renal or mesenteric hemodynamics. The hindquarters vasodilator effect of GI 262570 was not inhibited by the beta2-adrenoceptor antagonist, ICI 118551 ((+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol hydrochloride), and was still apparent in the presence of the alpha-adrenoceptor antagonist, phentolamine. Neither the latter, nor antagonism of angiotensin (AT1) and endothelin (ETA and ETB) receptors unmasked vasodilator responses to GI 262570 in the renal or mesenteric vascular beds. In the presence of GI 262570, vasodilator responses to acetylcholine and vasoconstrictor responses to methoxamine were normal. Furthermore, the cardiovascular responses to nonselective nitric-oxide synthase inhibition were not influenced by GI 262570. Collectively, these results indicate that the vasodilator action of GI 262570 is specific to the hindquarters vascular bed (of those studied), does not involve alpha- or beta2-adrenoceptors, and is not associated with a change in basal or stimulated nitric oxide release.

Acute cardiovascular effects of sibutramine in conscious rats.

Sibutramine is a serotonin and norepinephrine reuptake inhibitor, used in the treatment of obesity. In this study, cardiovascular effects of sibutramine (0.9, 3, or 9 mg kg(-1) i.p.) were measured in conscious Sprague-Dawley rats, in the absence and presence of beta- and/or alpha-adrenoceptor antagonism (with propranolol and/or phentolamine, respectively). Sibutramine caused pressor and tachycardic effects, with celiac and mesenteric vasoconstrictions, and hyperemic hindquarters vasodilatation. Pretreatment with propranolol inhibited the tachycardic and hindquarters vasodilator effect of sibutramine, whereas phentolamine inhibited the pressor and vasoconstrictor effects of sibutramine. In the presence of phentolamine, sibutramine caused hyperemic mesenteric vasodilatation. In preconstricted, isolated, mesenteric vessels, sibutramine and its metabolites BTS 54505 (N-desmethylsibutramine) and BTS 54354 (N-didesmethylsibutramine) (10 microM) produced significant vasodilations. Neither sibutramine nor BTS 54505 enhanced vessel sensitivity to norepinephrine, whereas BTS 54 354 produced a significant leftward shift in the concentration-response curve to norepinephrine. Collectively, the results indicate that the overt cardiovascular effects of sibutramine involve alpha-adrenoceptor-mediated celiac and mesenteric vasoconstrictions, and beta-adrenoceptor-mediated hindquarters vasodilatation and tachycardia. The mesenteric vasodilator response to sibutramine, seen in the presence of phentolamine, may be a direct effect of the drug and/or its metabolites, on vessel tone. The cardiovascular effects of sibutramine in vivo may be secondary to inhibition of peripheral and/or central reuptake of monoamines by the metabolites BTS 54354 and/or BTS 54505. It remains to explain why BTS 54354, but not BTS 54505, enhanced norepinephrine sensitivity in vitro, because both metabolites are potent inhibitors of the norepinephrine transporter.