ACE inhibitors and angiotensin II receptor antagonists.

The biological actions of angiotensin II (ANG), the most prominent hormone of the renin-angiotensin-aldosterone system (RAAS), may promote the development of atherosclerosis in many ways. ANG aggravates hypertension, metabolic syndrome, and endothelial dysfunction, and thereby constitutes a major risk factor for cardiovascular disease. The formation of atherosclerotic lesions involves local uptake, synthesis and oxidation of lipids, inflammation, as well as cellular migration and proliferation--mechanisms that may all be enhanced by ANG via its AT1 receptor. ANG may also increase the risk of acute thrombosis by destabilizing atherosclerotic plaques and enhancing the activity of thrombocytes and coagulation. After myocardial infarction, ANG promotes myocardial remodeling and fibrosis, and its many pathological mechanisms deteriorate the prognosis of these high-risk patients in particular. Therapeutically, inhibitors of the angiotensin I-converting enzyme (ACEI) and AT1 receptor blockers (ARB) are available to suppress the generation and cellular signaling of ANG, respectively. Despite major differences in the efficacy of ANG suppression and the modulation of other hormones and receptors, both classes of drugs are generally effective in attenuating numerous pathomechanisms of ANG in vitro, and in diminishing the development of atherosclerotic lesions and restenosis after angioplasty in various animal models. In clinical therapy, ACEI and ACE are well-tolerated antihypertensive drugs that also improve the prognosis of heart failure patients. After myocardial infarction and in stable coronary heart disease, ACEI have been shown to reduce mortality in a manner independent of hemodynamic alterations. However, there is little evidence that inhibitors of the RAAS may be effective against arterial restenosis, and a possible benefit of these substances compared to other antihypertensive drugs in the primary prevention of coronary heart disease in hypertensive patients is still a matter of debate, possibly depending on the specific substance and condition being investigated. As such, the general clinical efficacy of ACEI and ARB may be due to a positive influence on hemodynamic load, vascular function, myocardial remodeling, and neuro-humoral regulation, rather than to a direct attenuation of the atherosclerotic process. Further therapeutic advances may be achieved by identifying optimum drugs, patient populations, and treatment protocols.

Prepro-orexin and orexin receptor mRNAs are differentially expressed in peripheral tissues of male and female rats.

Orexins are produced specifically by neurons located in the lateral hypothalamus. Recent results suggested peripheral actions of orexins. Therefore, we analyzed the mRNA expression of prepro-orexin and the orexin receptor subtypes OX(1) and OX(2) in peripheral rat tissues. Using real-time quantitative RT-PCR we detected significant amounts of prepro-orexin mRNA in testis, but not in ovaries. OX(1) receptor mRNA was highly expressed in the brain and at lower levels in the pituitary gland. Only small amounts of OX(1) receptor mRNA were found in other tissues such as kidney, adrenal, thyroid, testis, ovaries, and jejunum. Very high levels of OX(2) receptor mRNA, 4-fold higher than in brain, were found in adrenal glands of male rats. Low amounts of OX(2) receptor mRNA were present in lung and pituitary. In adrenal glands, OX(2) receptor mRNA was localized in the zona glomerulosa and reticularis by in situ hybridization, indicating a role in adrenal steroid synthesis and/or release. OX(1) receptor mRNA in the pituitary and OX(2) receptor mRNA in the adrenal gland were much higher in male than in female rats. In the hypothalamus, OX(1) receptor mRNA was slightly elevated in female rats. The differential mRNA expression of orexin receptor subtypes in peripheral organs indicates discrete peripheral effects of orexins and the existence of a peripheral orexin system. This is supported by the detection of orexin A in rat plasma. Moreover, the sexually dimorphic expression of OX(1) and OX(2) receptors in the hypothalamus, pituitary, and adrenal glands suggests gender-specific roles of orexins in the control of endocrine functions.

Angiotensin II increases norepinephrine release from atria by acting on angiotensin subtype 1 receptors.

Norepinephrine stores in electrically driven guinea pig isolated atria were loaded with [3H]norepinephrine, and norepinephrine release was deduced from the radioactivity efflux. Electrical field stimulation of sympathetic nerve endings was applied during the refractory period of atrial contractions. The stimulation-induced release of norepinephrine was increased by angiotensin II (Ang II) (10(-8) to 10(-6) mol/L) in a concentration-dependent manner. The maximum observed effect was a 55% augmentation. The effects of 10(-7) and 10(-6) mol/L Ang II were abolished by 10(-6) and 10(-5) mol/L of the subtype 1 Ang II receptor antagonist losartan, respectively. Losartan by itself (10(-6) mol/L) caused a 14% reduction of norepinephrine release. The subtype 2 Ang II receptor ligand PD 123319 (1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)- 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate) in a concentration of 10(-4) mol/L had no detectable influence on transmitter release and did not antagonize the effect of Ang II. Angiotensin I (10(-6) and 10(-5) mol/L) increased norepinephrine release maximally by 23%. This effect was antagonized by 10(-5) mol/L losartan and did not appear in the presence of 10(-6) mol/L of the converting enzyme inhibitor ramiprilat. These results suggest that Ang II increases norepinephrine release by an activation of subtype 1 receptors, whereas angiotensin I is converted to Ang II to become effective.

Potentiation of the vascular response to kinins by inhibition of myocardial kininases.

Inhibitors of angiotensin I-converting enzyme (ACE) are very efficacious in the potentiation of the actions of bradykinin (BK) and are able to provoke a B(2) receptor-mediated vasodilation even after desensitization of this receptor. Because this activity cannot be easily explained only by an inhibition of kinin degradation, direct interactions of ACE inhibitors with the B(2) receptor or its signal transduction have been hypothesized. To clarify the significance of degradation-independent potentiation, we studied the vasodilatory effects of BK and 2 degradation-resistant B(2) receptor agonists in the isolated rat heart, a model in which ACE and aminopeptidase P (APP) contribute equally to the degradation of BK. Coronary vasodilation to BK and to a peptidic (B6014) and a nonpeptidic (FR190997) degradation-resistant B(2) agonist was assessed in the presence or absence of the ACE inhibitor ramiprilat, the APP inhibitor mercaptoethanol, or both. Ramiprilat or mercaptoethanol induced leftward shifts in the BK dose-response curve (EC(50)=3.4 nmol/L) by a factor of 4.6 or 4.9, respectively. Combined inhibition of ACE and APP reduced the EC(50) of BK to 0.18 nmol/L (ie, by a factor of 19) but potentiated the activity of B6014 (EC(50)=1.9 nmol/L) only weakly without altering that of FR190997 (EC(50)=0.34 nmol/L). Desensitization of B(2) receptors was induced by the administration of BK (0.2 micromol/L) or FR190997 (0.1 micromol/L) for 30 minutes; the vascular reactivity to ramiprilat or increasing doses of BK was tested thereafter. After desensitization with BK, but not FR190997, an additional application of ramiprilat provoked a B(2) receptor-mediated vasodilation. High BK concentrations were still effective at the desensitized receptor. The process of desensitization was not altered by ramiprilat. These results show that in this model, all potentiating actions of ACE inhibitors on kinin-induced vasodilation are exclusively related to the reduction in BK breakdown and are equivalently provoked by APP inhibition. The desensitization of B(2) receptors is overcome by increasing BK concentrations, either directly or through the inhibition of ACE. These observations do not suggest any direct interactions of ACE inhibitors with the B(2) receptor or its signal transduction but point to a very high activity of BK degradation in the vicinity of the B(2) receptor in combination with a stimulation-dependent reduction in receptor affinity.

Losartan attenuates symptomatic and hormonal responses to hypoglycemia in humans.

OBJECTIVE: Reduced awareness of hypoglycemic symptoms and compromised hormonal counterregulation increase the risk of severe hypoglycemia in people with diabetes mellitus. Up to the present, angiotensin 1 receptor blockers, which play an important role in controlling diabetic complications, have not been known to increase the risk of hypoglycemia. Nevertheless, we observed 3 cases of diabetic patients complaining of reduced awareness of hypoglycemic symptoms while they were under treatment with losartan in our outpatients clinic. We therefore investigated the effects of losartan on symptomatic and hormonal responses to hypoglycemia in humans. RESEARCH DESIGN AND METHODS: We carried out a randomized, double-blind, crossover study including 16 healthy men. The subjects received losartan 50 mg/d versus placebo. Treatment periods lasted for 7 days and were followed by a stepwise hypoglycemic clamp session (4.5 to 3.8 to 3.1 to 2.4 mmol/L) with measurement of counterregulatory hormones (epinephrine, norepinephrine, adrenocorticotropin, cortisol, glucagon), symptoms, and hemodynamic parameters (blood pressure, heart rate). RESULTS: Losartan attenuated the hypoglycemia-induced rise in plasma epinephrine (6480 +/- 490 pmol/L versus placebo 8970 +/- 790 pmol/L; P <.001) and the rise in plasma adrenocorticotropin (21 +/- 2 pmol/L versus 26 +/- 3 pmol/L; P <.01). Losartan also reduced symptom scores during hypoglycemia (P <.05). CONCLUSION: We conclude that short-term treatment with losartan slightly attenuates symptomatic and hormonal responses to hypoglycemia. At present, for patients who are unaware of hypoglycemia and who require antihypertensive or nephroprotective treatment, we would recommend caution concerning treatment with losartan.

Centrally bradykinin B2-receptor-induced hypertensive and positive chronotropic effects are mediated via activation of the sympathetic nervous system.

OBJECTIVE: The presence of bradykinin B2 receptors in the cardiovascular regulatory centres of the brain indicates that increase in mean arterial pressure (MAP) and heart rate after intracerebroventricular (i.c.v.) injections of bradykinin is mediated via stimulation of sympathetic nervous system. METHODS: Adult Wistar- Kyoto (WKY) rats were instrumented chronically with an i.c.v. cannula, and the catheters were placed into the femoral artery and vein. Increasing doses of bradykinin (1 -300 pmol) were given i.c.v. and (i) MAP and heart rate, (ii) plasma dopamine, noradrenaline and adrenaline, and (iii) plasma arginine vasopressin (AVP) levels were determined. In addition, following blockade of peripheral alpha1 -adrenoceptors with prazosin (50 and 250 microg/kg i.v.) beta1-adrenoceptors with atenolol (10 mg/kg i.v.) or V1 -receptors with TMe-AVP (Manning compound) (10 microg/kg i.c.v. and 100 microg/kg i.v.) the effects of bradykinin (100 pmol i.c.v.) on MAP and heart rate were determined. RESULTS: Bradykinin increased MAP and heart rate dose-dependently. The pressor effects of 100 pmol bradykinin i.c.v. were completely blocked by pretreatment with the specific B2 receptor antagonist Hoe 140 (3 pmol, i.c.v.). There was no change in plasma dopamine, noradrenaline, adrenaline or AVP levels after increasing doses of bradykinin. However, peripheral blockade of alpha1- and beta1-adrenoceptors reduced the bradykinin-induced increase in MAP and heart rate, whereas central and peripheral V1 receptor blockade did not alter the cardiovascular responses to i.c.v. bradykinin. CONCLUSION: Our data suggest that the hypertensive and positive chronotropic effects induced by i.c.v. bradykinin are due to stimulation of sympathoneuronal rather than sympathoadrenal pathway in vivo.

Angiotensin converting enzyme inhibition improves cardiac neuronal uptake of noradrenaline in spontaneously hypertensive rats.

OBJECTIVES: It has been shown that a diminished sympathetic activity contributes to the hypotensive and cardioprotective actions of angiotensin converting enzyme (ACE) inhibitors (ACEI). Besides an inhibition of central sympathetic tone and peripheral noradrenaline release, we hypothesized that the interactions of ACEI with the sympathetic system may include a modulation of neuronal catecholamine uptake by peripheral nerves. DESIGN: We investigated the influence of fosinopril on noradrenergic uptake into cardiac neurones in vitro and in vivo in acute and chronic models. METHODS AND RESULTS: Acute administration of fosinoprilat to isolated perfused rat hearts increased the extraction of [3H]-noradrenaline from the perfusate by 39%. Treatment (14 days) of spontaneously hypertensive rats (SHR) with fosinopril (20 mg/kg per day) enhanced the cardiac uptake of i.v. administered [3H]-noradrenaline by 28%. The endogenous left ventricular content of noradrenaline was increased by 49% after an antihypertensive treatment of SHR with fosinopril (20 mg/kg per day). Identical increases in cardiac noradrenaline stores (53%) were observed in SHR treated with a blood pressure ineffective dose of fosinopril (0.2 mg/kg per day). The myocardial content of adrenaline was increased in parallel to noradrenaline after both dose regimes. CONCLUSIONS: It is concluded that ACEI increases neuronal uptake of catecholamines in SHR in a blood pressure-independent manner. This effect occurs acutely and is independent of central sympathetic activity. Therefore, we hypothesize that ACEI modulate the activity of the cardiac noradrenaline transporter by direct activation. The improved uptake of noradrenaline may contribute to the antihypertensive and cardioprotective effects of ACEI.

Effects of angiotensin converting enzyme inhibitor on renal haemodynamics during mental stress.

OBJECTIVE: To examine the effects of angiotensin converting enzyme (ACE) inhibition on renal and systemic haemodynamics as well as on humoral regulators, under resting conditions and during mental stress in 20 normotensive and 20 mildly hypertensive subjects. METHODS: All of the subjects received either 25 mg cilazapril or placebo once a day, in a randomized, double-blind, cross-over trial for 1 week, followed by a 2-week washout period before the alternative regimen was given. We measured renal blood flow with para-aminohippuran, glomerular filtration rate with inulin, cardiac output by impedance cardiography and blood pressure and heart rate by an oscillometric method. We also monitored effects on plasma renin activity, aldosterone, catecholamines and atrial natriuretic peptide. Mental stress consisted of a long-lasting, time-reaction device, thereby provoking activation of the sympathetic nervous system. RESULTS: At rest ACE inhibition lowered mean arterial pressure (92 +/- 10 versus 98 +/- 9 mmHg), increased renal blood flow (803 +/- 109 versus 707 +/- 93 ml/min) and the renal fraction of cardiac output (25.9 +/- 2.5 versus 23.5 +/- 2.5%) and decreased the filtration fraction (17.9 +/- 2.5 versus 19.8 +/- 2.7%) in hypertensive but not in normotensive subjects. Sympathetic activation by mental stress leading to a transient increase in blood pressure did not alter significantly the effects of ACE inhibition on renal and systemic haemodynamics, in normotensive or in hypertensive subjects, although a tendency towards attenuation of the rise in glomerular filtration rate was noted in hypertensives (7.2 +/- 1.0 versus 5.1 +/- 0.8%). ACE inhibition led to increased plasma noradrenaline at rest but not during mental stress in hypertensive patients. CONCLUSION: ACE inhibition in patients with mild hypertension increased selectively renal perfusion, which is conserved during mental stress without persistent effects on the sympathetic nervous system. Thus, mental stress as a correlate of daily life stress appeared not to confound the selective renal vasodilatory effect of ACE inhibitors.

Effects of intracoronary low-dose enalaprilat as an adjunct to primary percutaneous transluminal coronary angiography in acute myocardial infarction.

Bradykinin accumulation is a potent cardioprotective mechanism underlying angiotensin-converting enzyme (ACE) inhibition in ischemia and/or reperfusion injury. There is, however, concern about treatment with ACE inhibitors in the very early phase of acute myocardial infarction (AMI) due to adverse systemic hemodynamic effects. We tested the hypothesis that cardiac bradykinin metabolism can be influenced by very low doses of intracoronary ACE inhibitors without harmful systemic effects in patients with AMI. Twenty-two patients with AMI in Killip classes II to III who underwent primary percutaneous transluminal coronary angiography (PTCA) were randomized to intracoronary enalaprilat (50 microg) or saline, given immediately after reopening of the infarct-related artery. Hemodynamics and electrocardiograms were monitored continuously and samples for determination of ACE activity, angiotensin II, bradykinin, kininogen, and cardiac marker proteins were collected from pulmonary arterial and central venous blood. Enalaprilat had no adverse effects on systemic hemodynamics, but rather stabilized arterial pressure and cardiac rhythm during reperfusion. Enalaprilat induced a 70% reduction of ACE activity and a significant increase of bradykinin in pulmonary arterial blood. Angiotensin II was not significantly affected by enalaprilat either in pulmonary arterial or in central venous blood. Myoglobin release was lower and the duration of reperfusion arrhythmias was significantly reduced in the enalaprilat group (p <0.05). Thus, in this pilot study, intracoronary enalaprilat infusion in the infarct-related artery is feasible in the setting of primary angioplasty and is safe and well tolerated. Effective cardiac ACE inhibition can be achieved by low-dose intracoronary enalaprilat, which primarily causes a potentiation of bradykinin.

Degradation of bradykinin by bovine tracheal epithelium and isolated epithelial cells.

1. The degradation of bradykinin (BK) labelled with tritiated proline at positions 2 and 3 ([3H]-BK) was determined on the luminal surface of bovine tracheal epithelium, in supernatants obtained from incubations of the luminal tracheal surface, and in suspensions of isolated tracheal epithelial cells. Peptidase inhibitors and identification of peptide fragments were used for characterization of the metabolic pathways. 2. On the luminal surface of intact bovine trachea, [3H]-BK was degraded with a half life of 12.8 min. [1-7]-BK and [1-5]-BK were the major direct metabolites which were further degraded via [1-3]-BK and [2-3]-BK to proline. Metabolism of [3H]-BK was unaltered in the presence of ramiprilat (250 nM) or phosphoramidon (10 microM). Phenanthroline diminished the formation of [1-7]- and [1-5]-BK and abolished the generation of proline. 3. Supernatants obtained from incubations of tracheal epithelium contained kininase activities which steadily increased when tracheae were incubated for longer than 30 min. After 60 min contact with epithelium, the incubation medium contained higher kininase activities than the epithelium itself. The spectrum of kinin metabolites generated by kininases in the supernatant was comparable to that formed by intact epithelium. 4. In suspensions of isolated epithelial cells, [3H]-BK was degraded with a half life of 70 min. The metabolites [1-3]- and [2-3]-BK were formed in parallel to [1-7]- and [1-5]-BK; however, proline was not generated. Degradation of [3H]-BK was not influenced by ramiprilat, but was inhibited by 85% in the presence of phosphoramidon. Phosporamidon markedly inhibited the generation of [1-7]- and [1-5]-BK and nearly abolished the formation of [1-3]- and [2-3]-BK. 5. In conclusion, angiotensin I-converting enzyme and neutral endopeptidase 24.11 are not significantly involved in [3H]-BK degradation on the luminal side of intact tracheal epithelium. The spectrum of metabolites found may in fact reflect the combined activities of metalloendopeptidase 24.15 and post-proline cleaving enzymes. Enzymes showing similar kininase activities are also released from the epithelium. Isolated epithelial cells contain low activities of these kininases, but a high activity of neutral endopeptidases, which may reflect an exclusively basolateral localization of the latter.

Intravascular and interstitial degradation of bradykinin in isolated perfused rat heart.

1. Bradykinin (BK) has been shown to exert cardioprotective effects which are potentiated by inhibitors of angiotensin I-converting enzyme (ACE). In order to clarify the significance of ACE within the whole spectrum of myocardial kininases we investigated BK degradation in the isolated rat heart. 2. Tritiated BK (3H-BK) or unlabelled BK was either repeatedly perfused through the heart, or applied as an intracoronary bolus allowing determination of its elution kinetics. BK metabolites were analysed by HPLC. Kininases were identified by ramiprilat, phosphoramidon, diprotin A and 2-mercaptoethanol or apstatin as specific inhibitors of ACE, neutral endopeptidase 24.11 (NEP), dipeptidylaminopeptidase IV and aminopeptidase P (APP), respectively. 3. In sequential perfusion passages, 3H-BK concentrations in the perfusate decreased by 39% during each passage. Ramiprilat reduced the rate of 3H-BK breakdown by 54% and nearly abolished [1-5]-BK generation. The ramiprilat-resistant kininase activity was for the most part inhibited by the selective APP inhibitor apstatin (IC50 0.9 microM). BK cleavage by APP yielded the intermediate product [2-9]-BK, which was rapidly metabolized to [4-9]-BK by dipeptidylaminopeptidase IV. 4. After bolus injection of 3H-BK, 10% of the applied radioactivity were protractedly eluted, indicating the distribution of this fraction into the myocardial interstitium. In samples of such interstitial perfusate fractions, 3H-BK was extensively (by 92%) degraded, essentially by ACE and APP. The ramiprilat- and mercaptoethanol-resistant fraction of interstitial kininase activity amounted to 14%, about half of which could be attributed to NEP. Only the product of NEP, [1-7]-BK, was continuously generated during the presence of 3H-BK in the interstitium. 5. ACE and APP are located at the endothelium and represent the predominant kininases of rat myocardium. Both enzymes form a metabolic barrier for the extravasated fraction of BK. Thus, only interstitial, but not intravascular concentrations of BK are increased by kininase inhibitors to the extent that a significant potentiation of BK effects could be explained. NEP contributes less than 5% to the total kininase activity, but is the only enzyme which is exclusively present in the interstitial space.

Apstatin, a selective inhibitor of aminopeptidase P, reduces myocardial infarct size by a kinin-dependent pathway.

1. Inhibitors of the angiotensin converting enzyme (ACE) have been shown to exert their cardioprotective actions through a kinin-dependent mechanism. ACE is not the only kinin degrading enzyme in the rat heart. 2. Since aminopeptidase P (APP) has been shown to participate in myocardial kinin metabolism to the same extent as ACE, the aims of the present study were to investigate whether (a) inhibition of APP leads to a reduction of myocardial infarct size in a rat model of acute ischaemia and reperfusion, (b) reduction of infarct size is mediated by bradykinin, and (c) a combination of APP and ACE inhibition leads to a more pronounced effect than APP inhibition alone. 3. Pentobarbital-anaesthetized rats were subjected to 30 min left coronary artery occlusion followed by 3 h reperfusion. The APP inhibitor apstatin, the ACE-inhibitor ramiprilat, or their combination were administered 5 min before ischaemia. Rats receiving HOE140, a specific B(2) receptor antagonist, were pretreated 5 min prior to enzyme inhibitors. Myocardial infarct size (IS) was determined by tetrazolium staining and expressed as percentage of the area at risk (AAR). 4. IS/AAR% was significantly reduced in rats that received apstatin (18+/-2%), ramiprilat (18+/-3%), or apstatin plus ramiprilat (20+/-4%) as compared with those receiving saline (40+/-2%), HOE (43+/-3%) or apstatin plus HOE140 (49+/-4%). 5. Apstatin reduces IS in an in vivo model of acute myocardial ischaemia and reperfusion to the same extent than ramiprilat. Cardioprotection achieved by this selective inhibitor of APP is mediated by bradykinin. Combined inhibition of APP and ACE did not result in a more pronounced reduction of IS than APP-inhibition alone.

Synthesis of kininogen and degradation of bradykinin by PC12 cells.

1. In this study, the abilities of PC12 cells to synthesize and degrade kinins were investigated. Kinin formation was assessed as kinin and kininogen content of cells and supernatants in serum-free incubations by use of a bradykinin-specific radioimmunoassay. Expression of kininogen mRNA was demonstrated by reverse-transcriptase PCR. Kinin degradation pathways of intact PC12 cells were characterized by identification of the kinin fragments generated from tritiated bradykinin either in the absence or presence of the angiotensin I-converting enzyme inhibitor ramiprilat. 2. Kinin immunoreactivity in the supernatant of PC12 cell cultures accumulated in a time-dependent fashion during incubations in serum-free media. This effect was solely due to de novo synthesis and release of kininogen (35 pg bradykinin h-1 mg-1 protein) since it could be suppressed by cycloheximide. Continuous synthesis of kininogen was a specific property of PC12 cells, as it was not observed in cultured macro- or microvascular endothelial cells. PC12 cells contained only minor amounts of stored kininogen. The rate of kininogen synthesis was not affected by ramiprilat, bacterial lipopolysaccharide, nerve growth factor or dexamethasone, but was stimulated 1.4 fold when cells were pretreated for 1 day with 1 microM desoxycorticosterone. 3. By use of cDNA probes specific for kininogen subtype mRNAs, expression of low-molecular-weight kininogen and T-kininogen in PC12 cells was confirmed. Expression of high molecular weight kininogen mRNA was also shown, though only at the lowest limit of detection of the assay. 4. Degradation of tritiated bradykinin by PC12 cells occurred with a half-life of 48 min resulting in the main fragments [1-7]- and [1-5]-bradykinin. The degradation rate of bradykinin decreased to 15% in the presence of ramiprilat (250 nM). Apart from angiotensin I-converting enzyme direct cleavage of bradykinin to [1-7]- and [1-5]-bradykinin still occurred under this condition as a result of additional kininase activities. 5. Along with previous findings of B2-receptor-mediated catecholamine release, these results now confirm the hypothesis that a cellular kinin system is expressed in PC12 cells. The presence of such a system may reflect a role of kinins as local neuromodulatory mediators in the peripheral sympathetic system.

Angiotensin converting enzyme inhibition by captopril influences cardiac work in healthy hearts.

Although beneficial effects of angiotensin converting enzyme (ACE) inhibition have been demonstrated in ill (ischemic, failing) hearts, it has not been proved that ACE inhibition induces changes in healthy hearts. The question is of clinical relevance, as many hypertensive patients do not display cardiac damage at the onset of treatment with ACE inhibitors, and possible changes in cardiac work might turn out more or less advantageous in the development of hypertensive heart disease. In a refined working heart preparation allowing measurement of cardiac work, including the contribution of atrial work and paracrine cardiac regulation, effects of captopril on cardiac dynamics were assessed. Coronary overflow of bradykinin, norepinephrine, and lactate was measured. Hearts were perfused for 20 min with vehicle or captopril at 3 x 10(-8), 3 x 10(-7), 3 x 10(-6), and 3 x 10(-5) mol/L. At the highest concentration, captopril increased coronary flow. Extending previous studies, the present study demonstrates that, in a concentration-dependent manner, captopril decreased oxygen consumption and maximal left ventricular pressure although the bradykinin outflow was not affected. From these influences of the drug on cardiac work and metabolism in healthy hearts, a protective influence of captopril in acute, critical situations of cardiac malnourishment or cardiac overload may be derived.

Radiation-induced heart disease: morphology, changes in catecholamine synthesis and content, beta-adrenoceptor density, and hemodynamic function in an experimental model.

To study further the pathophysiology of radiation-induced cardiomyopathy, we investigated resting hemodynamics, myocardial catecholamine synthesis and storage, and beta-adrenoceptor density after local heart irradiation. In Wistar rats, a radiation dose of 20 Gy eventually leads to compromised myocardial function which is characterized by a reduction in cardiac output to 43 +/- 11% and in the left ventricular ejection fraction to 66 +/- 7.5%, and an increase in the left ventricular end-diastolic volume to 187 +/- 17% of control values. This reduction in function is correlated with focal degeneration of 23 +/- 4% of the myocardium. Measurement of tyrosine hydroxylase activity and catecholamine content revealed that catecholamine biosynthesis is unchanged in the adrenals but is significantly reduced in the hearts of irradiated animals, while cardiac beta-adrenoceptor density is increased to about 140% of that in age-matched controls. This is in contrast to findings in dilated or ischemic cardiomyopathy. Time-course studies showed that the development of myocardial degeneration starts simultaneously with the decrease in cardiac output and ejection fraction and the increase in beta-adrenoceptors at 50-80 days postirradiation. Myocardial degeneration is maximal in extent and severity at 100 days and does not progress thereafter. Cardiac output decreases at 80-100 days postirradiation to 60 +/- 7% of control values. A significant further decrease is seen only when congestive heart failure becomes manifest at 249 +/- 21 days after 20 Gy. Thus there is a delay between structural myocardial injury and hemodynamic deterioration which could be due to a compensatory increase in beta-adrenoceptor density during the initial stages of the cardiomyopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Imidazoline binding sites on PC12 cells and bovine chromaffin cells.

Stimulating actions of imidazolines on the adrenal medulla were demonstrated by several laboratories. As only a few data about signal transduction exist, the aim of the present study is to establish a cellular model on which both subtypes of imidazoline receptors are present. Binding studies using [3H]clonidine for I1-sites and [3H]idazoxan for I2-sites were performed on bovine chromaffin cells and a PC12 cell line. The intracellular calcium signal was determined by a Fura-2 signal using a fluorescence microscope. Both subtypes of imidazoline binding sites are present on either crude membrane fractions, purified plasma membranes, and mitochondrial membranes of the adrenal medulla. Although the density of I1- and I2-sites on the plasma membrane fraction is almost equal, on the mitochondrial membrane fraction Bmax of I2-binding sites was double that of the I1-binding number. An increase in intracellular calcium signal could be obtained during stimulation of chromaffin cells by various I1- and I2-receptor modulators. Because a saturation of I2-binding could not be obtained in PC12 cells, adrenal medullary chromaffin cells may be a more suitable model for investigating imidazoline receptor signal transduction.

Modulation of MAO activity by imidazoline and guanidine derivatives.

I2-binding sites (I2-BS) are attributed to be a regulative site on monoamine oxidase (MAO). The in vivo and in vitro effects of various imidazoline and guanidine derivatives on MAO activity and on mitochondrial respiration were studied. Substances with high affinity for I2-BS (antazoline, idazoxan, and cirazoline: IC50 = 20.3, 33.8, and 43.4 microM) had a stronger inhibitory effect on MAO activity than did I1-ligands (efaroxan, rilmenidine, clonidine, and moxonidine: IC50 = 277, 801, 1,224, and > 10,000 microM). Substances with the highest inhibitory effects were BDF8082 (IC50 = 1.7 microM) and 2-(2-benzofuranyl)-2-imidazoline (BFI; IC50 = 4.0 microM). The enzyme is inhibited noncompetitively and is reversible, because its activity is completely or partially restored after dialysis. Agmatine, the putative endogenous ligand for IBS, also decreased MAO activity (IC50 = 168 microM), whereas its precursor, L-arginine, and its metabolite, putrescine, had no effects. In vitro inhibition of MAO and mitochondrial respiration by the IBS-ligands tested could not be correlated, suggesting no link between the function of the inner and outer mitochondrial membrane. MAO activity in vivo was significantly reduced only by pargyline (-95%), BDF8082 (-68%), BFI (-43%), and 1-(m-chlorophenyl)-biguanide (-28%). Catecholamine content of livers obtained from animals treated with different IBS-ligands was consequently increased. In conclusion, the strong inhibitory effects of I2 selective imidazoline ligands confirm the existence of I2-BS as a regulatory site on MAO.

Influence of imidazolines on catecholamine release in pithed spontaneously hypertensive rats.

The actions of the imidazoline derivatives clonidine, moxonidine, and rilmenidine and of the recently discovered clonidine-displacing substance agmatine on stimulation-induced norepinephrine overflow and epinephrine release were studied in pithed spontaneously hypertensive rats. All three imidazolines dose-dependently decreased norepinephrine overflow and led to an increase in epinephrine release when the highest dose of each compound was injected. The alpha 2-adrenoceptor antagonist rauwolscine shifted the dose-response curves of plasma norepinephrine concentrations to higher levels. Agmatine did not change norepinephrine overflow but increased epinephrine release into plasma after the highest dose administered. It is concluded that the investigated imidazolines decrease norepinephrine overflow via presynaptic alpha 2-adrenoceptors, whereas epinephrine release is mediated through putative imidazoline receptors on the adrenal medulla.

Orexin (hypocretin) gene expression in rat ependymal cells.

The expression of prepro-orexin (PPO) mRNA in the rat brain was investigated by in situ hybridization histochemistry. In the lateral and posterior hypothalamic areas, which are considered to produce exclusively PPO mRNA, we found high levels of PPO mRNA expressions. We also localized PPO mRNA hybridization signals at lower levels around the lateral ventricles, the third and fourth ventricle. Cellular analysis by emulsion autoradiography revealed the expression of PPO mRNA in the ependymal cell layer. Our results demonstrate that beside the lateral and posterior hypothalamus PPO mRNA is expressed in ependymal cells.

Sympathoadrenal dysfunction in rats with chronic neurogenic hypertension.

Compared to sham-operated controls 5 weeks after surgery neurogenic hypertensive rats with sino-aortic baroreceptor deafferentation had higher blood pressure, higher plasma noradrenaline and adrenaline levels, lower heart noradrenaline concentrations, higher adrenomedullary adrenaline levels and increased cardiac intraventricular pressure (dp/dtmax).