Effect of urotensin II on skin microvessel tone in diabetic patients without heart failure or essential hypertension.

Urotensin II (UII) is a potent vasoconstrictor peptide. Increased plasma levels and kidney expression of UII and its receptor have been observed in diabetes mellitus (DM). The aim of the present study was to evaluate the direct effect of exogenous UII on microvascular tone in DM patients compared with healthy controls. Vasoactive effect of UII (10(-12), 10(-9) and 10(-7) mol/L) on skin microvascular tone was evaluated in 12 controls and 12 DM patients (Type 1 or Type 2) without concomitant heart failure or essential hypertension using the non-invasive technique of iontophoresis and laser Doppler velocimetry. In addition, responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were evaluated. Urotensin II dose-dependently dilated skin microvasculature in control subjects (-51.8 +/- 59.4, 138.6 +/- 101.5, 204.2 +/- 115.7 and 207.5 +/- 81.6 arbitrary flux units (AFUs) for MilliQ and 10(-12), 10(-9) and 10(-7) mol/L UII, respectively) and dose-dependently vasoconstricted the microvasculature in DM patients (100.8 +/- 81.2, 46.2 +/- 85.1, 35.4 +/- 81.4 and 26.6 +/- 79.6 AFUs for MilliQ and 10(-12), 10(-9) and 10(-7) mol/L UII, respectively). Blood flow in control subjects and DM patients was differed significantly, with pair-wise comparisons indicating differences for 10(-9) and 10(-7) mol/L UII (P = 0.04 and P = 0.003). Results of blood flow in diet-controlled DM patients (204.7 +/- 193.6, 261.2 +/- 212.8, 256.1 +/- 202.9 and 233.7 +/- 115.9 AFUs for MilliQ and 10(-12), 10(-9) and 10(-7) mol/L UII, respectively) were similar to those in control subjects compared with results for DM patients receiving antidiabetic medication (48.8 + 80.0, -61.4 +/- 49.1, -75.0 +/- 40.0, -91.7 +/- 80.0 AFUs for MilliQ and 10(-12), 10(-9) and 10(-7) mol/L UII, respectively). Between-group significance remained after adjustment for baseline blood pressure values. Acetylcholine vasodilator responses were attenuated in DM patients compared with those in control subjects (1309.5 +/- 488.6 vs 3498.0 +/- 912.5 AFUs, respectively), whereas SNP responses were similar in the two groups (1467.9 +/- 411.3 vs 1984.4 +/- 410.7 AFUs, respectively). In conclusion, UII causes net vasoconstriction in DM. The UII-induced increases in peripheral vascular tone may contribute to DM-related cardiovascular complications.

Long-term but not short-term p38 mitogen-activated protein kinase inhibition improves cardiac function and reduces cardiac remodeling post-myocardial infarction.

p38 mitogen-activated protein kinase (p38 MAPK) inhibition exerts beneficial effects on left ventricular (LV) remodeling and dysfunction. p38 MAPK activity is transiently increased soon after myocardial infarction (MI), suggesting brief inhibition may afford the same benefit as long-term inhibition. We examined chronic 12-week p38 MAPK inhibition compared with short-term (7-day) inhibition, and then we discontinued inhibition after MI. Post-MI rats at day 7 received either vehicle, 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ67657; RWJ) for 12 weeks (long term; LT-RWJ), RWJ for 1 week and discontinued for 11 weeks (1-week RWJ), or continuous ramipril for 12 weeks. In separate groups of animals, 24 h after MI, vehicle or RWJ was administered for 7 days. Cardiac function was assessed by echocardiography and hemodynamic measurements. Percentage of fractional shortening improved after LT-RWJ and ramipril, but not after 1-week RWJ treatment. Likewise, LV contractility and maximal first derivative of left ventricular pressure (dP/dt(max)) was improved (12.5 and 14.4%) and LV end diastolic pressure (LVEDP) was reduced (49.4 and 54.6%) with both treatments. Functional outcomes were accompanied by regression of interstitial collagen I and alpha-smooth muscle actin expression in LV noninfarct, border, and infarct regions with LT-RWJ and ramipril treatment. Hypertrophy was reduced in noninfarct (18.3 and 12.2%) and border regions (16.3 and 12.0%) with both treatments, respectively. Animals receiving RWJ 24 h after MI for 7 days showed similar improvements in fractional shortening, dP/dt(max), LVEDP, including reduced fibrosis and hypertrophy. In vitro experiments confirmed a dose-dependent reduction in hypertrophy, with RWJ following tumor necrosis factor-alpha stimulation. Continuous but not short-term p38 MAPK blockade attenuates post-MI remodeling, which is associated with functional benefits on the myocardium.

Lidocaine and surgical modification reduces mortality in a rat model of cardiac failure induced by coronary artery ligation.

Coronary artery ligation in the rat provides a useful experimental model of cardiac failure; however, this procedure carries with it a high mortality rate (50%). In this study, we used lidocaine (10 mg/kg, i.m.) before coronary artery ligation and 2 h after surgery to minimise the incidence of ventricular fibrillation (VF) that leads to sudden death in this model. We found that coronary artery ligation, using lidocaine in conjunction with a modified surgical procedure, had a mortality rate of 15%, much lower than reported in previous studies using this model. These modifications allow for the production of larger infarcts with 29% of animals having an infarct size > 50% of the epicardial surface. Infarct size in our myocardial infarction (MI) group varied between 5% and 75% of the left ventricular (LV) surface area resulting in a mean infarct size of 41.3 +/- 1.3% for the epicardial surface and 40.0 +/- 1.3% for the endocardial surface.

Desensitization and resensitization of beta 1- and putative beta 4-adrenoceptor mediated responses occur in parallel in a rat model of cardiac failure.

1. Cardiostimulant effects of the non-conventional partial agonist, CGP 12177A, are mediated by a receptor distinct from the beta3-adrenoceptor and termed the putative beta4-adrenoceptor. Using a rat model of cardiac failure, induced by myocardial infarction (MI), we compared the desensitization and resensitization of responses to CGP 12177A with those to isoprenaline and RO 363 in left (LA) and right atria (RA). We also examined the ability of beta-adrenoceptor antagonists to block responses to CGP 12177A. 2. MI reduced the maximum inotropic response to isoprenaline by 48% (sham 4.1+/-0.6 mN, n=10; MI 2.1+/-0.4 mN, n=8, P<0.02), RO 363 by 61% (sham 4.2+/-0.5 mN, n=10; MI 1.8+/-0.3 mN, n=8, P<0.005) and CGP 12177A by 49% (sham 1.4+/-0.1 mN, n=5; MI 0.7+/-0.2 mN, n=7, P<0.05) in electrically stimulated LA. MI also reduced the sensitivity to isoprenaline (pEC50: sham 8.79+/-0.08, n=10; MI 8.30+/-0.10, n=8; P=0.001) and RO 363 (pEC50: sham 8.69+/-0.07, n=10; MI 8.33+/-0.10, n=8; P<0.01). The maximum chronotropic responses to isoprenaline, RO 363 and CGP 12177A in RA were unaffected. 3. Pertussis toxin treatment (10 microg kg-1, i.p.) restored the maximum inotropic response and sensitivity to isoprenaline (sham 3.5+/-0.5 mN, n=9; MI 3.2+/-0.6 mN, n=11, P=0.702) and CGP 12177A (sham 1.6+/-0.3 mN, n=6; MI 1.9+/-0.4 mN, n=7, P=0.537) in MI animals to levels similar to those in the sham group. 4. CGP 20712A (pKB: LA 6.7+/-0.2, n=6; RA 7. 1+/-0.1, n=4), ICI 118,551 (pKB: LA 6.4+/-0.1, n=5; RA 6.3+/-0.1, n=6), propranolol (pKB: LA 6.6+/-0.1, n=5; RA 6.8+/-0.1, n=6) and bupranolol (pKB: LA 7.2+/-0.1, n=6; RA 7.7+/-0.1, n=8), showed moderate affinity for the putative beta4-adrenoceptor. 5. Desensitization after MI and resensitization (after pertussis toxin treatment) to isoprenaline and CGP 12177A therefore occur in parallel, suggesting that the beta1- and putative beta4-adrenoceptor use the same signalling pathway. Antagonist affinity studies confirmed that drugs acting at beta1-adrenoceptors also interact with putative beta4-adrenoceptors with approximately 100 times lower affinity. We suggest that CGP 12177A produces its cardiac effects by interacting with a low affinity state of the beta1-adrenoceptor.

Desensitization of cardiac beta-adrenoceptor signaling with heart failure produced by myocardial infarction in the rat. Evidence for the role of Gi but not Gs or phosphorylating proteins.

This study examined mechanisms of beta-adrenergic (AR) desensitization in a myocardial infarction (MI) model of heart failure in the rat. Inotropic responses to isoproterenol (non-selective beta-AR agonist) and RO 363 (selective beta1-AR agonist), in left atria and left papillary muscle, were reduced by up to 65%, while chronotropic responses in right atria were unaffected. beta1- and beta2-AR density did not change after MI, suggesting that changes in beta-AR responsiveness are due to changes occurring downstream of the receptor. Inotropic and chronotropic responses to forskolin were not altered in right and left atria and left papillary muscle after MI, suggesting changes at the level of the G-proteins. Pertussis toxin treatment of animals restored inotropic responses to isoproterenol in left atria and left papillary muscle to levels seen in the sham group, indicating that inactivation of Gi-proteins improves inotropic function in MI rats, and that beta-ARs couple to Gi in cardiac failure. Expression of G-protein receptor kinase 2 (GRK2), beta-arrestin1 and the regulatory subunits of cAMPdPK (RI alpha and RII alpha), showed no change after MI. However the expression of Gi alpha2 was significantly increased in left ventricle (sham 0.888+/-0.140, MI 1. 759+/-0.352 P=0.026), right ventricle (sham 0.031+/-0.004, MI 0. 037+/-0.002 P=0.006) and atria (sham 0.107+/-0.006, MI 0.138+/-0.006 P=0.004), with no changes observed in the expression of Gs alpha. These results suggest that increases in Gi play an important role in the decreased beta-AR responsiveness in the rat model of MI.

Regulation of beta-adrenoceptors in a rat model of cardiac failure: effect of perindopril.

This study investigated the effects of myocardial infarction (MI)-induced cardiac failure and treatment with an angiotensin-converting enzyme (ACE) inhibitor perindopril (2 mg/kg/day) on rat beta-adrenoceptor (beta-ar) subtypes in anatomically defined regions of infarcted left ventricular (LV) free wall and noninfarcted tissue from right ventricle (RV) by using autoradiography. After 5 weeks of MI, rats with large MI size (>42%) had developed cardiac failure and beta1-ars were significantly decreased (-59%; p < 0.01) in the border region of the infarcted LV and almost abolished in the infarcted area (-90%; p < 0.005) compared with normal LV from sham-operated controls. The beta-ar changes were not found in the noninfarcted area of the same LV or in RV. MI did not significantly alter the number of beta2-ar subtypes in any region of the ventricles. Perindopril treatment for 4 weeks reduced mean cardiac region weights but did not affect beta-ar density in any cardiac region in either sham-operated or MI rats. These results indicate that cardiac failure due to MI causes significant downregulation of beta1-ars only in border and infarcted regions of rat LV and no change in beta2-ar in any area. It also suggests that the improved response of the infarcted rat heart to isoprenaline stimulation after ACE inhibitors does not result from changes in the numbers of cardiac beta-ars.

Signalling pathways in cardiac failure.

UNLABELLED: 1. Cardiac failure in humans and in animal models is associated with a marked desensitization of the catecholamine signalling pathway. 2. Beta 1- and beta 2- and possibly beta 3-adrenoceptors (beta-AR) are found in the hearts of humans and common laboratory animals such as rats and guinea-pigs. In rats and guinea-pigs chronic stimulation of cardiac beta-AR leads to a rapid loss of beta 2-AR whereas heart failure in humans is associated with a loss of beta 1-AR or beta 1-AR and beta 2-AR. 3. Desensitization is also associated with phosphorylation of beta-AR by beta-AR kinase (beta-ARK) and uncoupling of receptors from the signalling pathway. Beta-ARK but not beta-arrestin activity and mRNA are markedly increased in heart failure. 4. Chronic beta-AR stimulation and heart failure are associated with increases in Gi alpha but little if any change in Gs alpha. 5. The roles of beta gamma subunits of G-proteins, adenylate cyclase subtypes and cAMP dependent protein kinase A in heart failure are unclear at present. ABBREVIATIONS: beta-ARK - beta-adrenoceptor kinase AR - adrenoceptor G-protein - GTP binding protein

Beta-adrenoceptor regulation and functional responses in the guinea-pig following chronic administration of the long-acting beta 2-adrenoceptor agonist formoterol.

Formoterol is a long acting beta 2-adrenoceptor agonist designed for the alleviation of the symptoms of asthma. This study examined the effects of 14 day administration of formoterol (200 micrograms/kg/day i.p.) on beta 1- and beta 2-adrenoceptors in guinea-pig cardiac and lung tissue. Quantitative autoradiography was used to measure changes in receptor density and organ bath studies determined alterations in functional response. Formoterol treatment produced marked reductions of between 43% and 77% in beta 2-adrenoceptor density in all regions of the heart (atrioventricular node, bundle of His, right and left bundle branches, interventricular and interatrial septa, right and left atria, ventricles and apex) and lung (bronchial and vascular smooth muscle and parenchyma) (P < 0.01, n = 6). beta 1-Adrenoceptor density remained unchanged in all cardiac and lung regions. In functional studies (-)-isoprenaline was 4 fold less potent at causing relaxation of carbachol (1 microM) precontracted tracheal smooth muscle (pD2: control 8.49 +/- 0.03, formoterol 7.91 +/- 0.10, P < 0.001, n = 4), but formoterol treatment did not change the ability of (-)-isoprenaline to elicit a maximum response. The pKB values for ICI 118,551, 7.33 +/- 0.08 in the control and 7.20 +/- 0.01 in formoterol treated animals, were between those expected for beta 1- and beta 2-adrenoceptors suggesting involvement of both subtypes in the response. In spontaneously beating right atria and electrically paced left atria, tissues in which responses are largely mediated by beta 1-adrenoceptors, there was no significant change in responses to (-)-isoprenaline (right atria pD2: control 8.45 +/- 0.02; formoterol 8.42 +/- 0.11; P = 0.77, n = 4) (left atria pD2: control 8.25 +/- 0.03; formoterol 8.47 +/- 0.08; P = 0.09, n = 4). In the presence of CGP 20712A (100 nM) the pKB values did not change with formoterol treatment (left atria: control 9.59 +/- 0.12, formoterol 9.66 +/- 0.12; P = 0.70, n = 4) (right atria: control 8.93 +/- 0.11, formoterol 9.11 +/- 0.07; P = 0.25, n = 4). The doses and route of administration of formoterol used in this study differs from those used clinically. However, this study demonstrates that chronic formoterol administration produces selective down-regulation of beta 2-adrenoceptors in the lung and heart. The changes in the lung are accompanied by a shift to the right in the concentration-response curve to beta-agonist stimulation with no change in the maximum response.

Effect of chemical sympathectomy on (-)-isoprenaline-induced changes in cardiac beta-adrenoceptor subtypes in the guinea-pig and rat.

1. Quantitative autoradiography was used to determine beta-adrenoceptor densities in cardiac regions of guinea-pigs and rats after chemical sympathectomy with 6-hydroxydopamine, and to examine how chemical sympathectomy affected beta-adrenoceptor changes following infusion of (-)-isoprenaline (400 micrograms kg-1 hr-1, 7 days). 2. Seven days after 6-hydroxydopamine (100 mg kg-1, i.v.), cardiac tissue levels of noradrenaline were reduced by 94.0 +/- 3.5% (guinea-pig) and 86.0 +/- 7.0% (rat). The blood pressure increase in rats to tyramine (0.5 mg, i.v.) was reduced from 118 mmHg in controls to 4.4 mmHg in 6-hydroxydopamine-treated animals. 3. There were no changes 7 and 14 days after 6-hydroxydopamine treatment in total, beta 1-and beta 2-adrenoceptor density in the atrioventricular conducting system and atrial and ventricular myocardium in both species. 4. In control animals, (-)-isoprenaline infusion produced selective reductions in beta 2-adrenoceptor density, whilst beta 1-adrenoceptor density remained unchanged. 5. In 6-hydroxydopamine treated guinea-pigs or rats, (-)-isoprenaline infusion caused no change in beta 1-adrenoceptors except in the right bundle branch whilst beta 2-adrenoceptors were reduced in the atrioventricular conducting system (atrioventricular node, bundle of His, right and left bundle branches) and myocardium (interventricular septum and atria). 6. The differential effect of (-)-isoprenaline on beta 1- and beta 2-adrenoceptors is not therefore due to the occupation of beta 1-adrenoceptors by noradrenaline or to prior down-regulation of beta 1-adrenoceptors by noradrenaline, since it persists after depletion of noradrenaline.

Regulation of beta-adrenoceptors in the guinea-pig sinoatrial node.

This study examined the changes of beta-adrenoceptors in the guinea-pig sinoatrial nodal region following 7 day (-)-isoprenaline (400 micrograms/kg/h s.c.) infusion and the relationship between beta-adrenoceptor desensitization and receptor down-regulation. Changes in beta 1- and beta 2-adrenoceptor density were measured using quantitative autoradiography and function in organ bath studies. (-)-Isoprenaline treatment produced a marked decrease in total (from 57.5 to 33.9 fmol/mg protein), beta 1- (from 49.4 to 32.8 fmol/mg protein), and beta 2-adrenoceptor density (from 8.1 to 1.05 fmol/mg protein) in the sinoatrial node. In adjacent right atrium, treatment produced no change in total (39.5 and 36.7 fmol/mg protein) or beta 1-adrenoceptors (35.9 and 36.4 fmol/mg protein) but did decrease beta 2-adrenoceptors (from 3.7 to 0.3 fmol/mg protein). Chronotropic effects were measured in spontaneously beating right atrium. Procaterol, a selective beta 2-adrenoceptor agonist, caused a biphasic chronotropic response in control right atria, the first part of which was abolished in the tissue from treated animals. The maximum increase in right atrial rate to RO363, a beta 1-adrenoceptor selective partial agonist, was reduced from 114 bpm in control to 43 bpm in treated animals. In electrically driven right atrium with the sinoatrial node removed procaterol failed to produce a positive inotropic response via beta 2-adrenoceptors, but the maximum response to RO363 was reduced from 0.75 g in the control tissue to 0.12 g in the treated tissue. This study showed that changes in beta 2-adrenoceptor density following 7 day (-)-isoprenaline infusion are compatible with reduced functional responsiveness in the SA node. The reduction of beta 1-adrenoceptor number in the SA node was also compatible with the reduced chronotropic response in this tissue. However the lack of effect on beta 1-adrenoceptor density in the right atrium was not consistent with the decrease in beta 1-adrenoceptor mediated inotropic response in this tissue. This suggests that beta-adrenoceptor desensitization is not always associated with receptor down-regulation but depends also on the changes in the cell signalling system beyond the level of the receptor which differ according to the cardiac location.

Localization of (-)-[125I]cyanopindolol binding in guinea-pig heart: characteristics of non-beta-adrenoceptor related binding in cardiac pacemaker and conducting regions.

Receptor autoradiography was used in guinea-pig heart to locate binding sites for the beta-adrenoceptor ligand (-)[125I]cyanopindolol (CYP) resistant to blockade by the beta-adrenoceptor antagonist (-)-propranolol (1 microM). Highly localized binding was observed to regions closely associated with the sinoatrial node, atrioventricular node and bundle of His but was not observed on myocardial, pacemaker, conducting cells or adipose tissue. Free [125I] also bound to identical sites. Binding was enhanced in the presence of ascorbic acid but was completely inhibited by (-)-isoprenaline (100 microM), serotonin (5-HT) (10 microM) and phentolamine (10 microM).