'The effect of different genres of music on the stress levels of kennelled dogs'.

Classical music has been shown to reduce stress in kennelled dogs; however, rapid habituation of dogs to this form of auditory enrichment has also been demonstrated. The current study investigated the physiological and behavioural response of kennelled dogs (n=38) to medium-term (5days) auditory enrichment with five different genres of music including Soft Rock, Motown, Pop, Reggae and Classical, to determine whether increasing the variety of auditory stimulation reduces the level of habituation to auditory enrichment. Dogs were found to spend significantly more time lying and significantly less time standing when music was played, regardless of genre. There was no observable effect of music on barking, however, dogs were significantly (z=2.2, P<0.05) more likely to bark following cessation of auditory enrichment. Heart Rate Variability (HRV) was significantly higher, indicative of decreased stress, when dogs were played Soft Rock and Reggae, with a lesser effect observed when Motown, Pop and Classical genres were played. Relative to the silent period prior to auditory enrichment, urinary cortisol:creatanine (UCCR) values were significantly higher during Soft Rock (t=2.781, P<0.01) and the second silent control period following auditory enrichment (t=2.46, P<0.05). Despite the mixed response to different genres, the physiological and behavioural changes observed remained constant over the 5d of enrichment suggesting that the effect of habituation may be reduced by increasing the variety of auditory enrichment provided.

'Four Seasons' in an animal rescue centre; classical music reduces environmental stress in kennelled dogs.

On admission to rescue and rehoming centres dogs are faced with a variety of short- and long-term stressors including novelty, spatial/social restriction and increased noise levels. Animate and inanimate environmental enrichment techniques have been employed within the kennel environment in an attempt to minimise stress experienced by dogs. Previous studies have shown the potential physiological and psychological benefits of auditory stimulation, particularly classical music, within the kennel environment. This study determined the physiological/psychological changes that occur when kennelled dogs are exposed to long-term (7 days) auditory stimulation in the form of classical music through assessment of effects on heart rate variability (HRV), salivary cortisol and behaviour. The study utilised a cross over design in which two groups were exposed to two consecutive 7 day treatments; silence (control) and classical music (test). Group A was studied under silent conditions followed by 7 days of test conditions during which a fixed classical music playlist was played from 10:00-16:30 h. Group B received treatment in the reverse order. Results showed that auditory stimulation induced changes in HRV and behavioural data indicative of reduced stress levels in dogs in both groups (salivary cortisol data did not show any consistent patterns of change throughout the study). Specifically, there was a significant increase in HRV parameters such as µRR, STDRR, RMSSD, pNN50, RRTI, SD1 and SD2 and a significant decrease in µHR and LF/HF from the first day of silence (S1) to the first day of music (M1). Similarly, examination of behavioural data showed that dogs in both groups spent significantly more time sitting/lying and silent and less time standing and barking during auditory stimulation. General Regression Analysis (GRA) of the change in HRV parameters from S1 to M1 revealed that male dogs responded better to auditory stimulation relative to female. Interestingly, HRV and behavioural data collected on the seventh day of music (M2) was similar to that collected on S1 suggesting that the calming effects of music are lost within the 7 days of exposure. A small '9-Day' study was conducted in attempt to determine the time-scale in which dogs become habituated to classical music and examination of the results suggests that this occurs within as soon as the second day of exposure. The results of this study show the potential of auditory stimulation as a highly effective environmental enrichment technique for kennelled dogs. However, the results also indicate the requirement for further investigations into the way in which auditory stimulation should be incorporated within the daily kennel management regime in order to harness the full physiological and psychological benefits of music.

Investigation of the effects of a polymerised bovine haemoglobin solution on tension in isolated canine saphenous artery.

OBJECTIVES: To investigate the vasoconstriction induced by a polymerised bovine haemoglobin solution, Hb-200, in isolated canine arteries. METHODS: Rings of canine saphenous artery, from euthanatized dogs, were mounted between stainless steel wires in Krebs' solution (95% O2 , 5% CO2 , 37°C) for isometric tension recording. Following incubation with Hb-200, cumulative concentration response curves to phenylephrine (vasoconstrictor) and acetylcholine (vasodilator) were investigated. Responses to acute addition of Hb-200 were also examined in pre-constricted or pre-dilated arteries. Responses were further studied in the presence or absence of the endothelium, inhibitors of endothelium-dependent vasodilation (L-NAME, charybdotoxin and apamin), an endothelin antagonist (BQ-788) and the antioxidant superoxide dismutase. RESULTS: Incubation with Hb-200 (0·2 or 2 g/L) significantly enhanced phenylephrine-induced contraction (decreasing half maximal effective concentration, EC50 , P=0·0035) and inhibited acetylcholine-induced relaxation (increasing EC50 , P<0·0001). Acute addition of Hb-200 (0·2 or 2 g/L) significantly increased tension in pre-constricted arteries (P=0·0059) and reversed relaxation in pre-dilated arteries (P=0·0005). These acute responses were abolished in endothelium-denuded arteries and arteries incubated with L-NAME. Responses to Hb-200 were unaffected by incubation with charybdotoxin and apamin, BQ-788, or superoxide dismutase. CLINICAL SIGNIFICANCE: Low concentrations of Hb-200 enhance vasoconstriction in isolated canine saphenous artery, primarily by antagonism of nitric oxide. This effect may be detrimental in some dogs (e.g. those at risk of volume overload) but beneficial in others (e.g. those in septic shock).

Differential vasoactive effects of oestrogen, oestrogen receptor agonists and selective oestrogen receptor modulators in rat middle cerebral artery.

Cerebrovascular disorders are less common in pre-menopausal than post-menopausal women and in females than males. This protection may be due, in part at least, to direct effects of oestrogens on blood vessels. Oestrogen's vasodilatory mechanisms have been reported to be via the endothelium, vascular smooth muscle and extracellular matrix, depending on the vascular bed studied. Herein we investigated the vasoactive effects of oestrogen, oestrogen receptor (ER) and GPR30 agonists and selective ER modulators (SERMs) in the rat middle cerebral artery(MCA), an artery affected in focal ischaemia. MCAs isolated from male Sprague Dawley rats were mounted on a wire myograph. Concentration response curves were constructed to 17ß-oestradiol, ERα agonist-PPT, ERß agonist-DPN, GPR30 agonist-G1 and novel SERMs (LY362321 and LY2120310) in pre-constricted vessels, in the presence and absence of endothelium, blocking agents for nitric oxide synthase (L-NAME), classic ER antagonist (ICI182,780) or plasma membrane specific ERα (ERα-36) antibody. 17ß-oestradiol induced rapid vasorelaxation of the MCA which was not affected by endothelium removal, L-NAME or ICI182,780. Vasorelaxation was mimicked by PPT, DPN and G1 but not by the SERMs. Using ERα-36 antibody, effects of oestrogen were partially blocked. PPT had a greater vasorelaxation, while DPN and G1 had a lesser effect than 17ß-oestradiol. These findings indicate that activation of plasma membrane bound ERα, ß and GPR30 elicits rapid, endothelial-nitric oxide-independent relaxation of the rat MCA.

Neutering affects mRNA expression levels for the LH- and GnRH-receptors in the canine urinary bladder.

Neutering a bitch increases the incidence of acquired urinary incontinence (AUI) 20-fold. Mechanistically this effect is thought to be related to altered steroid/reproductive hormone concentrations and a recent study showed that gonadotrophin releasing hormone (GnRH) analogue treatment improved continence in bitches with AUI. The aim of this study was to examine mRNA expression levels for luteinizing hormone (LH)- and GnRH-receptors in the canine bladder and the correlation between these and in vitro contractility of the bladder using age matched entire and neutered, male and female canines and canines with AUI. Biopsies from the dome of the bladder were dissected post mortem with informed owner consent. mRNA expression for LH- and GnRH-receptor was quantified by rtPCR (relative to beta-actin). Contractility was assessed (cumulative concentration response curve for carbachol) in strips of bladder muscle using standard protocols. Analysis of variance (Tukey post-test) demonstrated thet neutering was associated with significantly increased levels of expression of LH- and GnRH-receptor mRNA in both sexes (P<0.01). mRNA expression for both receptors was significantly higher in female versus male canines. Neither effect was affected by animals' age and/or weight. A significant inverse correlation (Spearman's test) was found between bladder contractility and mRNA expression for both receptors. This effect was most pronounced in canines with AUI which demonstrated the highest mRNA expression levels yet had the lowest contractility of all animals studied. This suggests that increased LH- and GnRH-receptor mRNA expression is associated with changes in bladder function that increase an animal's predisposition to develop AUI.

Flow-induced enhancement of vasoconstriction and blockade of endothelium-derived hyperpolarizing factor (EDHF) by ascorbate in the rat mesentery.

BACKGROUND AND PURPOSE: We previously reported that ascorbate inhibits flow- and agonist-induced, EDHF-mediated vasodilatation in the bovine ciliary circulation. This study examined whether ascorbate had similar actions in the rat mesenteric vasculature. EXPERIMENTAL APPROACH: The effects of ascorbate were examined both in rat second order mesenteric arterial rings suspended in a static wire myograph and the rat mesentery perfused at different rates of flow. KEY RESULTS: Ascorbate (50 microM) had no effect on U46619-induced tone or acetylcholine-induced, EDHF-mediated vasodilatation in either rings of mesenteric artery or the perfused mesentery at rates of flow below 10 ml min(-1). At higher rates of flow, ascorbate produced two distinct effects in the rat mesentery: a rapid and maintained enhancement of vasoconstrictor tone and a slow (max at 3 h) inhibition of acetylcholine-induced, EDHF-mediated vasodilatation. The enhancement of vasoconstrictor tone appeared to be due to inhibition of flow-induced EDHF-like activity, since it was endothelium-dependent, but could be elicited during blockade of nitric oxide synthase and cyclooxygenase. Despite this, the classical inhibitors of EDHF, apamin and charybdotoxin, failed to affect the ascorbate-induced enhancement of tone, although they inhibited acetylcholine-induced vasodilatation. CONCLUSIONS AND IMPLICATIONS: Ascorbate inhibits both flow- and agonist-induced EDHF in the rat mesentery. The strikingly different timecourses of these two effects, together with their differential sensitivity to apamin and charybdotoxin, suggest that the flow- and agonist-induced EDHFs in the rat mesenteric vasculature may either be different entities or operate by different mechanisms.

Differential effects of glucose on agonist-induced relaxations in human mesenteric and subcutaneous arteries.

BACKGROUND AND PURPOSE: Acute periods of hyperglycaemia are strongly associated with vascular disorder, yet the specific effects of high glucose on human blood vessel function are not fully understood. In this study we (1) characterized the endothelial-dependent relaxation of two similarly sized but anatomically distinct human arteries to two different agonists and (2) determined how these responses are modified by acute exposure to high glucose. EXPERIMENTAL APPROACH: Ring segments of human mesenteric and subcutaneous arteries were mounted in a wire myograph. Relaxations to acetylcholine and bradykinin were determined in a control (5 mM) and high glucose (20 mM) environment over a 2 and 6 h incubation period. KEY RESULTS: Bradykinin-induced relaxation in both sets of vessels was mediated entirely by EDHF whilst that generated by acetylcholine, though principally generated by EDHF, also had contribution from prostacyclin and possibly nitric oxide in mesenteric and subcutaneous vessels, respectively. A 2-h incubation of high glucose impaired bradykinin-induced relaxation of subcutaneous vessels whilst, in contrast, the relaxation generated by bradykinin in mesenteric vessels was enhanced at the same time point. High glucose significantly augmented the relaxation generated by acetylcholine in mesenteric and subcutaneous vessels at a 2 and 6 h incubation point, respectively. CONCLUSIONS AND IMPLICATIONS: Short periods of high glucose exert a variable influence on endothelial function in human isolated blood vessels that is dependent on factors of time, agonist-used and vessel studied. This has implications for how we view the effects of acute hyperglycaemia found in patients with diabetes mellitus as well as other conditions.

The effect of the polymerized bovine haemoglobin solution, Hb-200, on endothelial function in isolated arterial rings from rats.

Haemoglobin-based oxygen carriers have been developed as 'blood substitutes'. Early cross-linked haemoglobins caused marked vasoconstriction, however, polymerized haemoglobin solutions, such as the bovine product haemoglobin glutamer-200 (bovine) (Hb-200), are said to have lesser vascular effects. The aim of this study was to quantify the effect of Hb-200 on isolated rat arterial rings. The actions of Hb-200 were investigated using rings of aorta and second-order mesenteric arteries from female Wistar rats. The arterial rings were mounted for isometric tension recording. Addition of Hb-200 (1 microM) to arterial rings, precontracted with phenylephrine (50-70% maximum phenylephrine-induced tone), resulted in vasoconstriction. Addition of Hb-200 to arteries precontracted with phenylephrine and relaxed with acetylcholine (1 microM) also caused contraction. Furthermore preincubation with Hb-200 (1 microM) enhanced the response to phenylephrine (1 nm to 10 microM) and impaired acetylcholine-induced relaxation (10 nm to 10 microM). Responses to Hb-200 were similar to those in response to the nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME, 100 microM), alone or in combination with Hb-200. These data demonstrate that Hb-200 has a significant vasoconstrictor effect similar to L-NAME. Thus the vascular actions of Hb-200 are consistent with activity as a NO scavenger. This may have implications for the clinical use of Hb-200.

Evidence for the expression and enzymatic activity of haem oxygenase-1 in the lungs of horses.

The aim of this study was to characterise the expression of haem oxygenase-1 (HO-1) in healthy lung tissue from horses and to measure its activity. Samples of lung tissue were collected from six horses euthanased for reasons other than respiratory disease. HO-1 expression and activity were detected in type II alveolar epithelial cells, macrophages and neutrophils in all the samples examined. The activity was dependent on the presence of NADPH and inhibited quantitatively by the addition of increasing concentrations of a competitive inhibitor of HO-1, tin mesoporphyrin IX.

Decreased basal despite enhanced agonist-stimulated effects of nitric oxide in 12-week-old stroke-prone spontaneously hypertensive rat.

This study examined both basal and agonist-stimulated effects of nitric oxide in rings of thoracic aorta and carotid artery from 12-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and compared them to those found in rings from normotensive Wistar Kyoto (WKY) controls. Acetylcholine-induced endothelium-dependent relaxation was found to be five-fold more sensitive in both male and female SHRSP when compared with those from age- and sex-matched WKY rats. In contrast, we found a reduction in the effects of basal nitric oxide in the SHRSP rat. Specifically, the ability of basal nitric oxide to depress contractile responses to phenylephrine was found to be reduced in vessels from SHRSP when compared with those from WKY rats. In addition, the endothelium-dependent depression of vasodilator responses to the nitric oxide donor, glyceryl trinitrate, was reduced in vessels from SHRSP when compared to those from WKY rats. Thus, we have shown that the effects of basal nitric oxide are impaired in the SHRSP rat at an age when the effects of agonist-stimulated nitric oxide are actually enhanced. This impairment may be related to the greater susceptibility of basal nitric oxide to destruction by superoxide anion which is known to be produced in excess in this model of hypertension.

Interaction between peroxynitrite and L-cysteine: effects on rat aorta.

In rings of rat aorta previously exposed to peroxynitrite (1 mM), L-cysteine and its analogues containing, but not those lacking, a thiol group produced a powerful transient relaxation. This relaxation is likely to result from the release of nitric oxide from a nitrated/nitrosated compound formed following reaction of peroxynitrite with a component of the tissue or bathing medium. Furthermore, when peroxynitrite was pre-mixed with L-cysteine a new relaxant species was formed. Analogues of L-cysteine with a free thiol reacted with peroxynitrite to form species with similar relaxant potencies. Analogues lacking a thiol formed products with relaxant activity, but less than with L-cysteine. Analogues with a free amino but no thiol or carboxylic functions formed products with potencies similar to those lacking only the thiol. If the amino is substituted and the thiol removed, no relaxant activity was generated. Thus, peroxynitrite reacts with L-cysteine to form a novel relaxant whose activity derives mainly from formation of its S-nitrosothiol, with a lesser component perhaps from an N-nitroso derivative.

Effects of chronic losartan treatment on vascular reactivity in normotensive rats.

OBJECTIVE: To investigate the vasoactive properties of large (aorta) and small (mesenteric) arteries in vitro after chronic losartan treatment of normotensive rats, hence providing information on the role played by angiotensin II in vascular tone. METHODS: Wistar rats were treated with 10 mg/kg per day losartan for 3 weeks. Ring segments of thoracic aorta and mesenteric resistance arteries (200 microns diameter) were mounted in myographs and wall force measured isometrically. RESULTS: The mean carotid blood pressure was reduced significantly after chronic losartan treatment (108 +/- 3 mmHg, n = 17 versus 116 +/- 2 mmHg, n = 16 in control rats, P < 0.05). In the mesenteric resistance artery the contractile response to 125 mmol/l K+, phenylephrine and angiotensin II was not affected significantly by losartan treatment. A subcontractile concentration of angiotensin II (0.1 nmol/l) induced a significant potentiation of the response to 0.03-100 mumol/l) phenylephrine (450 +/- 180 to 150 +/- 20% of the previous response to phenylephrine in control rats). This potentiation was attenuated significantly in the losartan group (240 +/- 80 to 100 +/- 15% of the previous response, P < 0.01 versus control rats). In the aorta, the response to 125 mmol/l K+ was not affected by chronic losartan treatment. The concentration required for the half-maximal effect for phenylephrine was increased significantly in the losartan group (0.51 +/- 0.11 mumol/l versus 0.17 +/- 0.03 mumol/l in controls rats; no change in maximum response) and the maximum response to angiotensin II was reduced significantly (0.7 +/- 0.08 mN/mg tissue versus 1.9 +/- 0.2 mN/mg tissue in control rats; the concentration for the half-maximal effect was not affected). Potentiation of phenylephrine-induced tone by 0.1 nmol/l angiotensin II (273 +/- 55 to 122 +/- 12% of the previous response in control rats) was attenuated significantly by losartan treatment (91 +/- 46 to 95 +/0 23% of the previous response, P < 0.01 versus control) CONCLUSIONS: Chronic administration of losartan could act on resistance arteries in normotensive rats by blocking the potentiation by angiotensin II of the agonist-induced tone.

Alteration of flow-induced dilatation in mesenteric resistance arteries of L-NAME treated rats and its partial association with induction of cyclo-oxygenase-2.

1. We investigated the response to pressure (myogenic tone) and flow of rat mesenteric resistance arteries cannulated in an arteriograph which allowed the measurement of intraluminal diameter for controlled pressures and flows. Rats were treated for 3 weeks with NG-nitro-L-arginine methyl ester (L-NAME, 50 mg kg-1 day-1) or L-NAME plus the angiotensin I converting enzyme inhibitor (ACEI) quinapril (10 mg kg-1 day-1). 2. Mean blood pressure increased significantly in chronic L-NAME-treated rats (155 +/- 4 mmHg, n = 8, vs control 121 +/- 6 mmHg, n = 10; P < 0.05). L-NAME-treated rats excreted significantly more dinor-6-keto prostaglandin F1 alpha (dinor-6-keto PGF1 alpha), the stable urinary metabolite of prostacyclin, than control rats. The ACEI prevented the rise in blood pressure and the rise in urinary dinor-6-keto PGF1 alpha due to L-NAME. 3. Isolated mesenteric resistance arteries, developed myogenic tone in response to stepwise increases in pressure (42 +/- 6 to 847 +/- 10 mN mm-1, from 25 to 150 mmHg, n = 9). Myogenic tone was not significantly affected by the chronic treatment with L-NAME or L-NAME + ACEI. 4. Flow (100 microliters min-1) significantly attenuated myogenic tone by 50 +/- 6% at 150 mmHg (n = 10). Flow-induced dilatation was significantly attenuated by chronic L-NAME to 22 +/- 6% at 150 mmHg (n = 10, p = 0.0001) and was not affected in the L-NAME + ACEI group. 5. Acute in vitro NG-nitro-L-arginine (L-NOARG, 10 microM) significantly decreased flow-induced dilation in control but not in L-NAME or L-NAME + ACEI rats. Both acute indomethacin (10 microM) and acute NS 398 (cyclo-oxygenase-2 (COX-2) inhibitor, 1 microM) did not change significantly flow-induced dilatation in controls but they both decreased flow-induced dilatation in the L-NAME and L-NAME + ACEI groups. Acute Hoe 140 (bradykinin receptor inhibitor, 1 microM) induced a significant contraction of the isolated mesenteric arteries which was the same in the 3 groups. 6. Immunofluorescence analysis of COX-2 showed that the enzyme was expressed in resistance mesenteric arteries in L-NAME and L-NAME + ACEI groups but not in control. COX-1 expression was identical in all 3 groups. 7. We conclude that chronic inhibition of nitric oxide synthesis is associated with a decreased flow-induced dilatation in resistance mesenteric arteries which was compensated by an overproduction of vasodilator prostaglandins resulting in part from COX-2 expression. The decrease in flow-induced dilatation was prevented by the ACEI, quinapril.

The effects of peroxynitrite on rat aorta: interaction with glucose and related substances.

Peroxynitrite (1-100 microM) induced a concentration-dependent relaxation of rat aortic rings; the logEC50 and maximum relaxation on endothelium-denuded rings were -5.31 +/- 0.03 and 105 +/- 5%, n = 6, respectively. The presence of the endothelium significantly impaired this relaxation (logEC50, -4.41 +/- 0.04; maximum relaxation, 71 +/- 4%; n = 6); an effect which was reversed by the inhibitor of nitric oxide synthase, N(G)-nitro-L-arginine methyl ester (L-NAME; 100 microM). Incubation with a high concentration of peroxynitrite (1 mM, 10 min followed by washout) had no effect on subsequent relaxation to acetylcholine (0.01-1 microM). It did, however, significantly depress subsequent contraction to phenylephrine (1-300 nM). This depression was dependent upon the presence of D-glucose in the Krebs solution, could be reversed by the inhibitor of soluble guanylate cyclase, methylene blue (10 microM) and reversed spontaneously after 2 h. When peroxynitrite (1 mM) was mixed with D-glucose (11 mM) and subsequently neutralised to remove unreacted peroxynitrite, a new more potent relaxant was formed. Despite this, the ability of peroxynitrite (1-100 microM) to produce relaxation of endothelium-denuded rings was similar in normal and glucose-free Krebs. Glycerol (22 mM), which like D-glucose is membrane permeant, also reacted with peroxynitrite (1 mM) to form a new more potent relaxant. L-cysteine (1 mM) had no effect by itself on the tone of aortic rings and when present in the tissue bath had no effect on the ability of peroxynitrite or neutralised peroxynitrite (1-100 microM) to produce relaxation. It did, however, potentiate the relaxant actions of the products formed from the reaction of peroxynitrite with D-glucose or glycerol. The membrane impermeant sugars, mannitol and sorbitol (each 11 mM) also reacted with peroxynitrite (1 mM), but expression of the vasorelaxant properties of their respective derivatives was seen only in the presence of L-cysteine (1 mM). Membrane permeance cannot, however, explain why peroxynitrite reacts with D-glucose and glycerol, but not mannitol or sorbitol to form products with intrinsic relaxant activity, as the product formed from the impermeant sugar, L-glucose (11 mM), also has intrinsic activity. The relaxant potency of this product was equipotent to that formed from D-glucose and was also potentiated by L-cysteine (1 mM). These result confirm that peroxynitrite can react with glucose and other compounds with alcohol functional groups to form vasorelaxant species. The relaxation induced when peroxynitrite is added to rat aortic rings is not, however, dependent upon this reaction since it occurs in glucose-free Krebs.

In vitro alteration of aortic vascular reactivity in hypertension induced by chronic NG-nitro-L-arginine methyl ester.

Chronic administration of NG-nitro-L-arginine methyl ester (L-NAME) induces a rise in blood pressure that is prevented by angiotensin I-converting enzyme inhibitors or angiotensin II receptor (type 1) blockade. Alterations in vascular reactivity in this model have not been extensively studied and could potentially be involved in the pathogenesis of L-NAME-induced hypertension. In the present work, we aimed to study the vascular reactivity and cGMP content of aortic ring segments isolated from Wistar rats treated for 3 weeks with L-NAME or L-NAME plus the converting enzyme inhibitor quinapril. Quinapril prevented the rise in blood pressure in L-NAME-treated rats although acetylcholine-induced dilation in aortic rings was suppressed and sodium nitroprusside-induced dilation was increased in both L-NAME- and L-NAME plus quinapril-treated rats. In isolated aortic ring segments, chronic L-NAME decreased the contractile response to K+ (125 mmol/L), phenylephrine, angiotensin II, the G protein stimulator AlF4-, and the protein kinase C activator phorbol dibutyrate. In contrast to the upregulated sodium nitroprusside-induced dilation, the contractile capacity of the aorta in response to angiotensin II, phenylephrine, AlF4-, K+, and phorbol dibutyrate was restored by quinapril. Aortic cGMP was lowered in rats treated with L-NAME (530 +/- 120 fmol/mg protein, n = 12, P < .05) and L-NAME plus quinapril (461 +/- 140 fmol/mg protein, n = 12, P < .05) compared with controls (1798 +/- 522 fmol/mg protein, n = 12). We hypothesize that the continuous nitric oxide blockade by L-NAME might attenuate a continuous endogenous relaxing tone and is associated with an upregulated endogenous vasoconstrictor tone in large arteries. Converting enzyme inhibition interfered more with the increased endogenous constrictor tone than with the decreased vasodilator tone in the wall of large arteries from L-NAME-treated rats.

Effects of manipulation of dietary cholesterol on the function of the thoracic aorta from New Zealand white rabbits.

Animal studies, while generally showing loss of endothelium-dependent responses after an elevation in plasma cholesterol, have provided conflicting reports with regard to recovery of function after normalisation of cholesterol level. Therefore, we assessed changes in vascular function after a period of hypercholesterolaemia and the subsequent effect of normalisation of cholesterol levels. Contractile responses to phenylephrine (PE) and endothelium-dependent relaxation in response to carbachol were examined in thoracic aorta from New Zealand White rabbits (NZW) fed a 0.3% cholesterol diet for 20 weeks, from NZW fed a 0.3% cholesterol diet for 20 weeks, followed by standard diet for 20 more weeks, and from their respective age-matched controls. Cholesterol levels were increased in rabbits receiving the 0.3% cholesterol diet (12.7 +/- 3.2 mM; 0.5 +/- 0.1 mM control) and returned to normal when standard diet was reintroduced (0.8 +/- 2.0 mM). Contractile responses were not affected by the period of hypercholesterolaemia. Carbachol-induced relaxation of a submaximal PE contraction was impaired after the period of hypercholesterolaemia (Emax 69 +/- 9%; 95 +/- 3% age-matched (control); the effect was reversed after reintroduction of standard diet (Emax 79 +/- 6%; 82 +/- 2% age-matched control). Our results demonstrate that endothelium-dependent relaxation is impaired after a long-term 0.3% cholesterol diet. Furthermore, after reintroduction of a normal diet, there is no further impairment of endothelium-dependent relaxation and endothelium function improves.

Chronic infusion of low-dose angiotensin II potentiates the adrenergic response in vivo.

OBJECTIVE: The aim of this study was to investigate the role of angiotensin II-induced potentiation of the alpha 1-adrenergic contractile response in the aetiology of low-dose angiotensin II-induced hypertension. METHODS: Wistar rats (250g) received angiotensin II (120 ng/kg per min) from subcutaneous minipumps for 21 days. The responses of vaso-active properties of second-order mesenteric arteries (200 micron) to potassium, phenylephrine, angiotensin II and acetylcholine were assessed. The acute amplification effects of angiotensin II on the response to phenylephrine were examined. RESULTS: Angiotensin II induced a progressive hypertension, which reached a plateau after approximately 5 days. The responses to potassium, angiotensin II and acetylcholine were not significantly modified in rats treated chronically with angiotensin II. The major finding of this study is that the response to phenylephrine (1-3 mumol/l) was potentiated (sevenfold at 1.75 mumol/l) after chronic treatment with angiotensin II. In control vessels acute addition of angiotensin II (10(-10) mol/l) produced no contraction but induced potentiation of the phenylephrine response (1-3 mumol/l). No further potentiation of the phenylephrine response was observed in the rats treated chronically with angiotensin II. CONCLUSIONS: Thus, although the direct contractile responses to potassium and angiotensin II remain unaffected following chronic angiotensin II treatment, the alpha 1-adrenergic contractile response to phenylephrine is significantly potentiated by angiotensin II in this model of hypertension. We suggest that this potentiation contributes to the hypertension observed in response to infusion of low-dose angiotensin II.

Vascular reactivity in mesenteric resistance arteries following chronic nitric oxide synthase inhibition in Wistar rats.

1. Chronic inhibition of nitric oxide synthase (NOS) induces a sustained hypertension in rats. We studied the effects of chronic inhibition on the in vitro vasoreactivity of mesenteric resistance arteries in Wistar rats. We also investigated the effects of acute in vitro NOS inhibition in these vessels. 2. Acute NOS inhibition (N omega-nitro-L-arginine, L-NOARG, 10 microM) had no effect on the contractile response to KCl (125 mM), enhanced the response to the phorbol ester, phorbol dibutyrate (1 microM; 69 +/- 9% of KCl response, n = 6; 38 +/- 7% control, n = 6, P < 0.05), increased sensitivity to phenylephrine (EC50: 1.68 +/- 0.14 microM, n = 5; 2.35 +/- 0.23 microM control, n = 5, P < 0.05) and sodium nitroprusside (SNP; EC50 1.79 +/- 0.61 nM, n = 6; 20.44 +/- 6.87 nM control, n = 6, P < 0.05) and decreased sensitivity to acetylcholine (EC50 123 +/- 12 nM, n = 6; 45 +/- 10 nM control, n = 13, P < 0.05). 3. In contrast, contractile responses to KCl (125 mM; 170 +/- 12 mN mm-3, n = 10; 257 +/- 21 mN mm-3 in control, n = 13, P < 0.005) and phenylephrine (maximum response, 30 microM: 169 +/- 24 mN mm-3, n = 10; 295 +/- 19 mN mm-3 in control, n = 13, P < 0.001) were significantly reduced in magnitude following chronic NOS inhibition. Sensitivity to phenylephrine was not significantly altered. 4. The effects of chronic NOS inhibition (N omega-nitro-L-arginine methyl ester, L-NAME, 10 mg kg-1 daily for 3 weeks) were similar to those of acute NOS blockade with respect to the relaxant responses to SNP and acetylcholine, and also the contraction in response to protein kinase C activation. 5. Chronic inhibition of NOS significantly increased medial cross sectional area of mesenteric resistance arteries (0.013 +/- 0.002 mm2, n = 7; 0.009 +/- 0.0005 mm2 control, n = 15, P < 0.05). 6. Thus, in contrast to the acute effects of NOS inhibition, chronic NOS inhibition results in a down-regulation of the contractile responses to KCl and phenylephrine in mesenteric resistance arteries, despite an increase in medial cross sectional area. However protein kinase C-dependent contraction remains relatively enhanced. Endothelium-dependent relaxation is reduced and endothelium-independent relaxation is enhanced in a manner similar to the effects of acute NOS blockade.

Development and progression of atherosclerosis in aorta from heterozygous and homozygous WHHL rabbits. Effects of simvastatin treatment.

This study was conducted to define progression of atherosclerosis in both homozygous and heterozygous Watanabe heritable hyperlipidemic (WHHL) rabbits and to investigate the ability of the HMG CoA reductase inhibitor simvastatin to attenuate progression of the disease. We examined contractile responses to phenylephrine and endothelium-dependent relaxation in response to carbachol in thoracic aorta at 3, 6, 9, and 12 months in control New Zealand White (NZW) rabbits, homozygous WHHL rabbits, and heterozygous WHHL rabbits. Homozygous and heterozygous rabbits were treated with simvastatin (10 mg/kg per day) from 3 to 6 months and from 9 to 12 months of age. Simvastatin significantly reduced serum cholesterol levels in young heterozygotes, with a nonsignificant trend toward a reduction in older heterozygotes. In homozygotes, no significant fall was observed. Contractile function declined progressively with age in all groups--most in homozygotes and least in NZW rabbits. Relaxation was unaffected by age in NZW rabbits; relaxation declined in the heterozygotes and declined to a greater extent in homozygotes. Simvastatin retarded the loss of function in the young heterozygotes. Similar trends were observed in young homozygotes and older heterozygotes, with no effect in older homozygotes. Histological studies revealed the progressive development of early atherosclerosis in heterozygotes, and more advanced atherosclerosis was observed in homozygotes. Simvastatin did not inhibit development of atheroma. A correlation was observed between vascular function and structure. However, functional changes preceded the development of atheroma. In addition, we have demonstrated that simvastatin can help to reduce the loss of vascular function associated with the progression of atherosclerosis in the heterozygous WHHL rabbit.

Effects of a xanthine oxidase/hypoxanthine free radical and reactive oxygen species generating system on endothelial function in New Zealand white rabbit aortic rings.

We investigated the effects of the xanthine oxidase (XO)/hypoxanthine (HX) free radical (FR) generating system on the relaxant properties of aortic rings from New Zealand White rabbits. This system generates superoxide anions, hydroxyl radicals, and H2O2. We wished to identify which of these species is responsible for impairment of vascular function. After obtaining dose-response curves to phenylephrine (PE) and carbachol or sodium nitroprusside (SNP), we exposed rings to the FR generating system or H2O2 for 30 min, either with or without a range of potentially protective agents. Dose-response curves to carbachol or SNP were then repeated. Exposure to the XO/HX system impaired endothelium-dependent, carbachol-induced relaxation. The hydroxyl radical scavengers mannitol, N-(2-mercaptopropionyl)-glycine (MPG), and captopril offered no protection. Superoxide dismutase (SOD) increased the impairment of response, catalase provided partial protection, and a combination of SOD and catalase completely prevented impairment of the response. H2O2 mimicked the effects of XO/HX system. H2O2 appears to be the primary species involved in mediating the toxic effects of the XO/HX FR generating system, but the superoxide anion is probably responsible for some of the loss of relaxation and a role for intracellular generation of hydroxyl radicals cannot be excluded.