Pertti Arvola

Exercise enhances vasorelaxation in experimental obesity associated hypertension

OBJECTIVE Regular exercise is recommended for the non-pharmacological treatment of hypertension, but the mechanisms underlying the lowering of blood pressure remain controversial. Therefore, we studied the effects of 22-week-long training on blood pressure, arterial reactivity, and metabolic abnormalities in a model of genetic obesity and moderate hypertension. METHODS Obese and lean Zucker rats were subjected to treadmill exercise from 8 to 30 weeks of age. Blood pressures were measured by the tail-cuff method, and urine was collected in metabolic cages. At the end of the study, the samples for biochemical determinations were taken, and reactivity of isolated mesenteric and carotid arterial rings was examined in standard organ chambers. RESULTS The exercise prevented the elevation of blood pressure which was observed in non-exercised obese Zucker rats, and also reduced blood pressure in the lean rats. The relaxations of norepinephrine-preconstricted mesenteric and carotid arterial rings to acetylcholine and nitroprusside were clearly improved by exercise in the obese rats. In the lean rats exercise enhanced vasorelaxation to nitroprusside in the mesenteric and carotid rings, and to acetylcholine in the carotid preparations. The exercise-induced improvement of endothelium-mediated dilatation to acetylcholine was abolished by nitric oxide synthesis inhibition with NG nitro-L-arginine methyl ester, but not by cyclooxygenase inhibition with diclofenac or functional inhibition of endothelium-dependent hyperpolarization by precontractions with KCl. The urinary excretion of the systemic prostacyclin metabolite (2,3-dinor-6-ketoprostaglandin F1 alpha) was increased two-fold by exercise in the obese and lean rats, whereas that of the thromboxane A2 metabolite (11-dehydrothromboxane B2) remained unaffected. Treadmill training reduced blood glucose, cholesterol, and triglycerides, but did not affect the high levels of insulin in obese Zucker rats. CONCLUSIONS These results suggest that the antihypertensive effect of long-term exercise in experimental obesity related hypertension is associated with improved vasodilatation. This is expressed as enhanced relaxation via endogenous and exogenous nitric oxide, and increased endothelial prostacyclin production. The improved control of arterial tone after training could be attributed to the alleviation of hyperlipidemia and insulin resistance, whereas hyperinsulinaemia per se remained unaffected.

Endothelial function in spontaneously hypertensive rats: influence of quinapril treatment

1 Angiotensin converting enzyme (ACE) inhibition has been shown to restore the impaired endothelial function in hypertension, but the mediators underlying the promoted endothelium‐dependent dilatation have not been fully characterized. Therefore, we investigated the effects of 10‐week‐long quinapril therapy (10 mg kg−1 day−1) on responses of mesenteric arterial rings in vitro from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto (WKY) rats. 2 Endothelium‐dependent relaxations of noradrenaline (NA)‐precontracted rings to acetylcholine (ACh) and adenosine 5′‐diphosphate (ADP) were similar in WKY rats and quinapril‐treated SHR and more pronounced than in untreated SHR. The nitric oxide (NO) synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) attenuated the relaxations in both WKY groups and quinapril‐treated SHR, and completely inhibited them in untreated SHR. When endothelium‐dependent hyperpolarization was prevented by precontraction of the preparations with potassium chloride (KC1), no differences were found in relaxations to ACh and ADP between the study groups. In addition, in NA‐precontracted rings the L‐NAME‐ and indomethacin‐resistant relaxations to ACh were partially prevented by apamin, an inhibitor of calcium‐activated potassium channels. 3 Interestingly, in quinapril‐treated SHR but not in the other groups, exogenous bradykinin potentiated the relaxations to ACh in both NA‐ and KCl‐precontracted arterial rings. 4 Contractile sensitivity of endothelium‐intact rings to NA was reduced in SHR by quinapril, and was more effectively increased by L‐NAME in quinapril‐treated than untreated SHR. 5 In conclusion, since the relaxations to ACh and ADP in quinapril‐treated SHR were augmented in the absence and presence of NO synthesis inhibition but not under conditions which prevented hyperpolarization, enhanced endothelium‐dependent relaxation after long‐term ACE inhibition can be attributed to increased endothelium‐dependent hyperpolarization. However, the potentiation of the response to ACh by exogenous bradykinin in quinapril‐treated SHR, as well as the increased attenuating effect of the endothelium on NA‐induced contractions in these animals appear to result from enhanced endothelium‐derived NO release.

Endothelial Function in Deoxycorticosterone-NaCl Hypertension Effect of Calcium Supplementation

BACKGROUND Dietary calcium intake has been suggested to correlate inversely with blood pressure in humans and experimental animals. However, the effects of calcium supplementation on hypertensive disturbances of the endothelium have not been well characterized. METHODS AND RESULTS Wistar-Kyoto rats made hypertensive by deoxycorticosterone (DOC)-NaCl treatment, but a concurrent increase in chow calcium content from 1.1% to 2.5% markedly attenuated the rise in blood pressure. The function of isolated mesenteric arterial rings in vitro was investigated at the close of the 10-week study. In norepinephrine-precontracted rings, the relaxations to acetylcholine (ACh) and ADP, as well as to nitroprusside, 3-morpholinosydnonimine, and isoproterenol were attenuated in hypertensive rats on 1.1% calcium supplementation. In the presence of NG-nitro-L-arginine methyl ester (L-NAME), the relaxations to ACh in hypertensive animals on normal calcium were practically absent, whereas in normotensive rats and calcium-supplemented hypertensive rats, distinct relaxations to higher concentrations of ACh were still present. These responses were reduced by 30% to 50% with apamin, a blocker of Ca2+-activated K+ channels, and were further inhibited by blockade of ATP-dependent K+ channels with glyburide. Interestingly, relaxations elicited by ACh and ADP during precontraction with 60 mmol/L KCl (preventing endothelium-dependent hyperpolarization) were not impaired in hypertensive animals. The contractile sensitivity of endothelium-intact arterial rings to 5-hydroxytryptamine and norepinephrine was higher in hypertensive rats on either normal or high-calcium diet, whereas the increase in contractile sensitivity caused by L-NAME corresponded in all groups. CONCLUSION High-calcium diet markedly opposed experimental DOC-NaCl hypertension, an effect associated with improved arterial relaxation, while abnormalities of vascular contractile properties remained unaffected. In particular, the hyperpolarization-related component of endothelium-dependent arterial relaxation, mediated via opening of arterial K+ channels, could be augmented by calcium supplementation in DOC-NaCl hypertension.

Effects of botulinum toxin A on the sphincter of Oddi: an in vivo and in vitro study

Background—Botulinum toxin A is a potent inhibitor of the release of acetylcholine from nerve endings. Local injection of botulinum toxin has recently been suggested to be helpful in sphincter of Oddi dyskinesia by decreasing sphincter of Oddi pressure. Aims—To explore the mechanism of action of botulinum toxin A on sphincter of Oddi (SO) muscle. Methods—Four piglets underwent duodenoscopy and SO manometry was performed. After obtaining a baseline pressure, the SO was injected with normal saline and the experiment repeated after one week. The SO was then injected endoscopically with botulinum toxin (40 U) with follow up manometry one week later. The sphincter of Oddi was removed from 10 pigs, cut into three rings, and placed in an organ bath. The force of contraction was measured and registered on a polygraph. Rings were stimulated by 70 V (10 Hz, 0.5 ms) electrical field stimulation for 20 seconds, exogenous acetylcholine (100 μM), and KCl (125 mM). Botulinum toxin (0.1 U/ml) or atropine (1 μM) was added to the incubation medium and the stimulation was repeated. Results—Mean basal SO pressure in the pigs remained unchanged after saline injection but decreased to about 50% of baseline value following botulinum toxin injection (p=0.04). The contractions induced by direct stimulation of SO smooth muscle with KCl were not significantly affected by either atropine or botulinum toxin. In all rings exogenous acetylcholine induced contractions, which were totally blocked by atropine, but not by botulinum toxin. Electrical field stimulation induced contractions that were inhibited by both atropine and botulinum toxin. Conclusion—Botulinum toxin inhibits pig sphincter of Oddi smooth muscle contractions by a presynaptic cholinergic mechanism, similar to that described in skeletal muscle.

Contractions induced by potassium‐free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats

1 Vascular contractions induced by K+‐free solution and relaxation responses following the return of K+ to the organ bath were studied in mesenteric arterial rings from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto rats (WKY) with particular focus on the role of vascular adrenergic nerve‐endings and endothelium. 2 In endothelium‐denuded rings the omission of K+ from the incubation medium resulted in gradual contractions, the rate of which was slower in SHR than WKY. Nifedipine (1 μm) inhibited the contractions more effectively in SHR than WKY. 3 Adrenergic denervation in vitro with 6‐hydroxydopamine reduced the contractions induced by the K+‐free medium in endothelium‐denuded rings. The remaining contractions after denervation were markedly greater in SHR than WKY. 4 The presence of intact vascular endothelium attenuated the K+‐free contractions in both strains, the attenuation being smaller in SHR than WKY. NG‐nitro‐l‐arginine methyl ester (l‐NAME, 0.1 mm) and methylene blue (10 μm), but not indomethacin (10 μm), abolished the attenuating effect of endothelium on the K+‐free contractions. l‐Arginine (1 mm) reversed the effect of l‐NAME in WKY but not in SHR. 5 The re‐addition of K+ after full K+‐free contractions dose‐dependently relaxed the rings. The rate of this K+‐induced relaxation was significantly slower in SHR than WKY at all K+ concentrations (0.1–5.9 mm) studied, whether the endothelium or functioning adrenergic nerve‐endings were present or not. Ouabain (1 mm) totally inhibited the K+ relaxation in SHR but only partially in WKY. 6 Vascular smooth muscle contractions induced by high concentrations of potassium were comparable between the strains. The EC50 for noradrenaline‐induced contractions was lower in SHR than WKY, but the maximal forces did not differ significantly. 7 In conclusion, the contractile response in K+‐free solution more clearly differentiates vascular rings from SHR and WKY than the responses induced by the classical contractile agents noradrenaline and high concentrations of potassium. The depressant effect of the presence of intact endothelium on the K+‐free contractions, which was smaller in SHR than WKY, is mediated via the endothelium‐derived relaxing factor. Neurotransmitter release from vascular adrenergic nerve‐endings participates less in the K+‐free contractile response in SHR than WKY. Moreover, the contractile response is more dependent on calcium entry through nifedipine‐sensitive calcium channels in SHR than WKY. The greater K+‐free contractions of denervated endothelium‐denuded rings and the reduced K+ relaxation rate in SHR when compared to WKY suggest increased cell membrane permeability and decreased activity of vascular Na+, K+‐ATPase, respectively, in this type of genetic hypertension.

Quinapril treatment and arterial smooth muscle responses in spontaneously hypertensive rats

1 The effects of long‐term angiotensin‐converting enzyme inhibition with quinapril on arterial function were studied in spontaneously hypertensive rats, Wistar‐Kyoto rats serving as normotensive controls. 2 Adult hypertensive animals were treated with quinapril (10 mg kg−1 day−1) for 15 weeks, which reduced their blood pressure and the concentrations of atrial natriuretic peptide in plasma and ventricular tissue to a level comparable with that in normotensive rats. 3 Responses of mesenteric arterial rings in vitro were examined at the end of the study. Compared with normotensive and untreated hypertensive rats, responses to noradrenaline were attenuated in hypertensive animals on quinapril, both force of contraction and sensitivity being reduced. Quinapril also attenuated maximal contractions but not sensitivity to potassium chloride. Nifedipine less effectively inhibited vascular contractions in normotensive and quinapril‐treated than in untreated hypertensive rats. 4 Arterial relaxation responses by endothelium‐dependent (acetylcholine) and endothelium‐independent (sodium nitrite, isoprenaline) mechanisms were similar in normotensive and quinapril‐treated rats and more pronounced than in untreated hypertensive rats. 5 Cell membrane permeability to ions was evaluated by means of potassium‐free solution‐induced contractions of endothelium‐denuded denervated arterial rings. These responses were comparable in normotensive and quinapril‐treated rats and less marked than in untreated hypertensive rats. 6 Intracellular free calcium concentrations in platelets and lymphocytes, measured by the fluorescent indicator quin‐2, were similar in normotensive and quinapril‐treated rats and lower than in untreated hypertensive rats. 7 In conclusion, quinapril treatment improved relaxation responses and attenuated contractions in arterial smooth muscle of hypertensive rats. These changes may be explained by diminished cytosolic free calcium concentration, reduced cell membrane permeability, and alterations in dihydropyridine‐sensitive calcium channels following long‐term angiotensin‐converting enzyme inhibition.

Dietary calcium and magnesium supplements in spontaneously hypertensive rats and isolated arterial reactivity

1 High calcium diet attenuates the development of hypertension but an associated undesirable effect is that Mg2+ loss to the urine is enhanced. Therefore, we studied the effects of high calcium diet alone and in combination with increased magnesium intake on blood pressure and arterial function. 2 Forty‐eight young spontaneously hypertensive rats (SHR) were allocated into four groups, the dietary contents of Ca2+ and Mg2+ being: 1.1%, 0.2% (SHR); 2.5%, 0.2% (Ca‐SHR); 2.5%, 0.8% (CaMg‐SHR); and 1.1%, 0.8% (Mg‐SHR), respectively. Development of hypertension was followed for 13 weeks, whereafter electrolyte balance, lymphocyte intracellular free calcium ([Ca2+]i), and mesenteric arterial responses in vitro were examined. Forty normotensive Wistar‐Kyoto (WKY) rats were investigated in a similar manner. 3 Calcium supplementation comparably attenuated the development of hypertension during normal and high magnesium intake in SHR, with an associated reduced lymphocyte [Ca2+]i and increased Mg2+ loss to the urine. 4 Endothelium‐dependent arterial relaxation to acetylcholine was augmented in Ca‐SHR and CaMg‐SHR, while the relaxations to isoprenaline and the nitric oxide donor SIN‐1 were similar in all SHR groups. Relaxation responses induced by the return of K+ to the organ bath upon precontractions in K+‐free solution were used to evaluate the function of arterial Na+, K+‐ATPase. The rate of potassium relaxation was similar in Ca‐SHR and CaMg‐SHR and faster than in untreated SHR. 5 Contractile responses to high concentrations of potassium and noradrenaline, and the ability of vascular smooth muscle to sequester Ca2+, which was evaluated by eliciting responses to caffeine or noradrenaline after loading periods in different Ca2+ concentrations, were comparable in all SHR groups. In SHR with increased magnesium intake, and in WKY rats with calcium or magnesium supplementation, no detectable effects on blood pressure and arterial function were observed. 6 In conclusion, high calcium diet attenuated the development of hypertension in SHR, with an associated augmented endothelium‐dependent relaxation, promoted recovery rate of ionic gradients across the cell membrane via Na+, K+‐ATPase, and reduced basal [Ca2+]i. Dietary magnesium supplementation, whether combined with normal or high calcium intake, had no beneficial effects on blood pressure or arterial function.

High calcium diet augments vascular potassium relaxation in hypertensive rats.

The effects of increased dietary calcium on the development of hypertension and vascular smooth muscle responses were studied in spontaneously hypertensive rats and normotensive Wistar-Kyoto rats. Both hypertensive and normotensive animals were divided into two groups; the calcium content of the normal diet was 1.1% and that of the high calcium diet 3.1%. During the 12-week study, calcium supplementation significantly attenuated the increase in systolic blood pressure in the hypertensive rats but did not affect blood pressure in the normotensive rats. The contractile responses of endothelium-denuded mesenteric arterial rings to potassium chloride were similar in all study groups. The contractions to norepinephrine were not altered by the high calcium diet either, but smooth muscle sensitivity to this agonist was lower in the normotensive than in the hypertensive rats. Potassium relaxation was used to evaluate the activity of vascular smooth muscle Na+,K(+)-ATPase. The maximal rate of potassium relaxation was fastest in the normotensive groups but was also clearly faster in calcium-treated hypertensive rats when compared with hypertensive rats on a normal diet. Platelets were used as a cell model for the analysis of intracellular free calcium concentration, which was measured by the fluorescent indicator quin-2. Intracellular free calcium was significantly reduced in the hypertensive rats by calcium supplementation and was not affected in the normotensive rats. In conclusion, a reduction of intracellular free calcium concentration indicating improved calcium regulation and a concomitant alteration in vascular relaxation probably reflecting increased activity of smooth muscle Na+,K(+)-ATPase may contribute to the blood pressure-lowering effect of a high calcium diet.

Arterial contractions induced by cumulative addition of calcium in hypertensive and normotensive rats: influence of endothelium

Responses to cumulative addition of Ca2+ (0.2–2.5 mM) after precontraction with potassium chloride (KCl) and noradrenaline in Ca2+-free medium were studied in isolated mesenteric arterial rings from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The Ca2+ contractions in 125 mM KCl-stimulated endothelium-denuded rings in the presence of atenolol (10 μM) and phentolamine (10 μM) were less marked in SHR than WKY, although the contractions to high concentrations of KCl in normal organ bath Ca2+ (1.6 mM) were similar in these strains. The difference in Ca2+ contractions between SHR and WKY during KCl stimulation was also present after 10-min pretreatment with 1 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) in Ca2+-free medium. However, when noradrenaline (1 μM) was used as the agonist the Ca2+ contractions of endothelium-denuded rings in the two strains were comparable, while exposure to EGTA reduced these responses more effectively in SHR than WKY. Nifedipine (0.5 nM and 10 nM in KCl- and noradrenaline-stimulated rings, respectively) more efficiently inhibited the Ca2+ contractions in hypertensive than in normotensive rats.The presence of intact vascular endothelium attenuated the contractions to Ca2+ addition comparably (during KCl stimulation) or even more (during noradrenaline) in SHR when compared with WKY NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM) counteracted this attenuation correspondingly in WKY and SHR, and L-arginine (1 mM) restored it in both strains, whereas indomethacin (10 mM) was without effect on the response. However, mesenteric arterial relaxations induced by the endothelium-dependent agonists acetylcholine and ADP in noradrenaline-precontracted (1 μM) rings were clearly impaired in SHR, and also L-NAME (0.1 mM) reduced the responses to acetylcholine more efficiently in SHR. In contrast, the relaxations to acetylcholine and ADP in KCl-precontracted (60 mM) rings in the absence and presence of L-NAME were comparable between the two strains.In conclusion, attenuated contractile response to cumulative Ca2+ addition during stimulation with KCl clearly differentiated arterial smooth muscle of hypertensive and normotensive rats, suggesting altered function of cell membrane in SHR. The more pronounced effect of nifedipine on the response indicates abnormal function of voltage-dependent Ca2+ channels, and higher diminishing effect of EGTA on the contraction during noradrenaline suggests exaggerated action of the chelator on membrane-bound Ca2+ in SHR. Interestingly, the depressant effect of intact endothelium on the Ca2+ contraction response, mediated largely via nitric oxide, was not attenuated in SHR. Furthermore, impaired endothelium-dependent agonist-induced relaxations can be attributed to reduced release of endothelium-derived hyperpolarizing factor in this type of genetic hypertension.

Direct Effect of Thyroxine on Pig Sphincter of Oddi Contractility

Sphincter of Oddi (SO) motility has an important role in the regulation of bile flow. SO function disturbances (stenosis or dyskinesia) may prevent normal bile flow and thus enhance the probability of common bile duct (CBD) stone formation. Previously we have shown that there is an increased prevalence of diagnosed hypothyroidism in CBD stone patients, compared with gallbladder stone patients or age-, sex-, and hospital-admission-adjusted controls. The present study was done to test the hypothesis that thyroxine directly effects the SO. The specificity of the effects of thyroxine were studied by comparing with triiodothyronine (T3), progesterone, cortisone, estrogen, and testosterone. For ex vivo studies three or four successive 1 to 1.5-mm SO rings were prepared from each pig and placed between two hooks in oxygenated physiologic salt solution at 37°C. SO contraction was measured with isometric force displacement transducers and registered on a polygraph. Each SO ring was stimulated with KCl (125 mM), acetylcholine (ACh; 10 or 100 μM) and histamine (Hist; 10 or 100 μM) with and without thyroxine (10-10 or 10 -8 M), T3 (10-9 or 10-7 M), progesterone (1 μM), cortisone (1 μM), estrogen (1 μM), or testosterone (1 nM) in the medium. KCl, ACh, and Hist induced strong contractions in the SO rings. The addition of thyroxine did not influence significantly the KCl-induced contractions, but the ACh- and Hist-induced contractions decreased by a mean of 37–44% (P < 0.001) and 54–56% (P < 0.001), respectively, as compared to the contractions without thyroxine. Triiodothyronine had a similar inhibitory effect to thyroxine, whereas cortisone, estrogen, and testosterone had no effect. Progesterone decreased the KCl-, ACh-, and Hist-induced SO contractions. In conclusion, physiological concentrations of thyroxine have an inhibitory effect on receptor-mediated ACh and Hist, but not on the nonspecific KCl-induced SO contraction ex vivo. The inhibitory effect is similar in thyroxine and triiodothyronine. Of the steroid hormones, only progesterone nonspecifically ameliorates SO contractions ex vivo. Because the effect of thyroxine on the SO is prorelaxing, the lack of thyroxine may result in an increased tension of the SO.