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This study sets out to characterise calcitonin gene-related peptide (CGRP) receptors in human and porcine isolated proximal and distal coronary arteries using BIBN4096BS. Human (h)-alphaCGRP induced relaxations that were blocked by BIBN4096BS in all arteries studied. In contrast to the other vessels, the Schild plot slope in the human distal coronary artery segments (0.68 +/- 0.07) was significantly less than unity and BIBN4096BS potently blocked these responses (pK(b) (10 nM): 9.29 +/- 0.34, n = 5). In the same preparation, h-alphaCGRP(8-37) behaved as a weak antagonist of h-alphaCGRP-induced relaxations (pK(b) (3 microM): 6.28 +/- 0.17, n = 4), with also a Schild plot slope smaller than unity. The linear agonists, [ethylamide-Cys(2,7)]-h-alphaCGRP ([Cys(Et)(2,7)]-h-alphaCGRP) and [acetimidomethyl-Cys(2,7)]-h-alphaCGRP ([Cys(Acm)(2,7)]-h-alphaCGRP), had a high potency (pEC(50): 8.21 +/- 0.25 and 7.25 +/- 0.14, respectively), suggesting the presence of CGRP(2) receptors, while the potent blockade by BIBN4096BS (pK(b) (10 nM): 10.13 +/- 0.29 and 9.95 +/- 0.11, respectively) points to the presence of CGRP(1) receptors. Using RT-PCR, mRNAs encoding for the essential components for functional CGRP(1) receptors were demonstrated in both human proximal and distal coronary artery. Further, h-alphaCGRP (100 nM) increased cAMP levels, and this was attenuated by BIBN4096BS (1 microM). The above results demonstrate the presence of CGRP(1) receptors in all coronary artery segments investigated, but the human distal coronary artery segments seem to have an additional population of CGRP receptors not complying with the currently classified CGRP(1) or CGRP(2) receptors.

Background Circulating angiotensin (Ang) II accumulates in adrenal tissue via binding to Ang II type 1 (AT1) receptors, reaching levels that are 15 to 20 times higher than in blood. Adrenal tissue contains a second renin transcript that gives rise to a truncated prorenin representing a cytosolic form of renin. Here we investigated what percentage of adrenal Ang II originates at adrenal tissue sites, and whether intracellular renin contributes to adrenal angiotensin production. Methods Concentrations of endogenous and iodine-125 (125I)–labeled Ang I and II were measured in adrenal tissue and blood from pigs after 125I-Ang I infusion. Results In the adrenal tissue in all animals, 125I-Ang I was undetectable. In untreated pigs, adrenal 125I-Ang II was 17 ± 1 times arterial 125I-Ang II, and tissue Ang I and II were 5 ± 1 and 388 ± 40 times higher than plasma Ang I and II. The AT1 receptor antagonist eprosartan reduced adrenal 125I-Ang II accumulation by 80%, and increased plasma Ang II to a greater degree than tissue Ang II. As a consequence, eprosartan equally reduced the tissue/plasma concentration ratios of both Ang II and 125I-Ang II. Captopril did not alter 125I-Ang II accumulation, and acutely, but not chronically, reduced the adrenal Ang II/I ratio. Conclusions More than 90% of adrenal Ang II originates at adrenal tissue sites. Local adrenal Ang II generation occurs extracellularly and is followed by internalization via AT1 receptor-mediated endocytosis. Enhanced angiotensin generation, combined with incomplete AT1 receptor blockade and the large adrenal AT1 receptor reserve, explains why eprosartan increased rather than decreased adrenal Ang II. Our data do not support angiotensin generation by truncated prorenin.

Background— Angiotensin (Ang) II type 2 (AT 2 ) receptor stimulation results in coronary vasodilation in the rat heart. In contrast, AT 2 receptor–mediated vasodilation could not be observed in large human coronary arteries. We studied Ang II–induced vasodilation of human coronary microarteries (HCMAs). Methods and Results— HCMAs (diameter, 160 to 500 μm) were obtained from 49 heart valve donors (age, 3 to 65 years). Ang II constricted HCMAs, mounted in Mulvany myographs, in a concentration-dependent manner (pEC 50 , 8.6±0.2; maximal effect [E max ], 79±13% of the contraction to 100 mmol/L K + ). The Ang II type 1 receptor antagonist irbesartan prevented this vasoconstriction, whereas the AT 2 receptor antagonist PD123319 increased E max to 97±14% ( P max was larger in older donors (correlation ΔE max versus age, r =0.47, P 2 ) receptor antagonist Hoe140, or after removal of the endothelium. Ang II relaxed U46619-preconstricted HCMAs in the presence of irbesartan by maximally 49±16%, and PD123319 prevented this relaxation. Finally, radioligand binding studies and reverse transcription–polymerase chain reaction confirmed the expression of AT 2 receptors in HCMAs. Conclusions— AT 2 receptor–mediated vasodilation in the human heart appears to be limited to coronary microarteries and is mediated by B 2 receptors and NO. Most likely, AT 2 receptors are located on endothelial cells, and their contribution increases with age.

To investigate the mediators of bradykinin-induced vasorelaxation in human coronary microarteries (HCMAs), HCMAs (diameter ≈300 μm) obtained from 42 heart valve donors (20 men and 22 women; age range, 3 to 65 years; mean age, 46 years) were mounted in Mulvany myographs. In the presence of the cyclooxygenase inhibitor indomethacin, bradykinin relaxed preconstricted HCMAs in a concentration-dependent manner. N G -nitro- l -arginine methyl ester and ODQ (inhibitors of nitric oxide [NO] synthase and guanylyl cyclase, respectively) and the NO scavenger hydroxocobalamin, either alone or in combination, shifted the bradykinin concentration-response curve to the right. Removal of H 2 O 2 (with catalase), inhibition of cytochrome P450 epoxygenase (with sulfaphenazole or clotrimazole) or gap junctions (with 18α-glycyrrhetinic acid or carbenoxolone), and blockade of large- (BK Ca ) and small- (SK Ca ) conductance Ca 2+ -dependent K + channels (with iberiotoxin and apamin), either alone or in addition to hydroxocobalamin, did not affect bradykinin. In contrast, complete blockade of bradykinin-induced relaxation was obtained when we combined the nonselective BK Ca and intermediate-conductance (IK Ca ) Ca 2+ -dependent K + channel blocker charybdotoxin and apamin with hydroxocobalamin. Charybdotoxin plus apamin alone were without effect. Inhibition of inwardly rectifying K + channels (K IR ) and Na + /K + -ATPase (with BaCl 2 and ouabain, respectively) shifted the bradykinin concentration-response curve 10-fold to the right but did not exert an additional effect in the presence of hydroxocobalamin. In conclusion, bradykinin-induced relaxation in HCMAs depends on (1) the activation of guanylyl cyclase, K IR , and Na + /K + -ATPase by NO and (2) IK Ca and SK Ca channels. The latter are activated by a factor other than NO. This factor is not a cytochrome P450 epoxygenase product or H 2 O 2 , nor does it depend on gap junctions or BK Ca channels.

Angiotensin II is a peptide hormone regulator of blood pressure and fluid balance in mammals. Evidence obtained largely in vitro has also suggested that angiotensin II has growth-promoting effects and that it might thereby contribute to such pathological phenomena as cardiac hypertrophy, a major risk factor for cardiovascular mortality. It has been difficult to test for the direct growth-promoting effects of angiotensin II in vivo, however, because of the generalized effects of the peptide on hemodynamics. To overcome this limitation and to test for cardiac-specific functions of angiotensin II, we generated transgenic mice expressing an angiotensin II-producing fusion protein exclusively in cardiac myocytes. Our findings are the first to distinguish between local and systemic effects of angiotensin II on the heart and introduce a novel technique for studying tissue-specific peptide function.

To investigate whether tissue angiotensin II generation occurs intra- or extracellularly, we studied the subcellular localization of angiotensin II in kidney and adrenal, two organs with high endogenous angiotensin II concentrations.Tissues were obtained, following a 1 h infusion of 125I-angiotensin I or 125I-angiotensin II to simultaneously determine the localization of plasma-derived angiotensin II, from five control pigs and four pigs that had been pretreated with the AT1 receptor antagonist eprosartan. Subcellular organelles, prepared by differential centrifugation from homogenized tissue, were characterized using organelle-specific markers.125I-angiotensin II and angiotensin II were present in all organelles, with identical distribution profiles. In mitochondria-enriched fractions the relative specific activities [RSAs = (concentration per mg protein in fraction)/(concentration per mg protein in homogenate)] of the two peptides were similar to those in homogenate, whereas in cytosol-enriched fractions their RSAs were five- to 10-fold lower (P0.05 versus homogenate). In microsome- as well as in lysosome-enriched fractions the RSAs of 125I-angiotensin II and angiotensin II were two- to four-fold higher than in homogenate (P0.05), and their RSAs were also higher in renal nuclei-enriched fractions (P0.05). Eprosartan increased plasma angiotensin II to a larger degree than tissue angiotensin II and greatly reduced tissue 125I-angiotensin II. This led to similar decreases in the tissue/plasma concentration ratios of 125I-angiotensin II and angiotensin II. The subcellular distribution of both angiotensin II peptides was not affected by eprosartan.Local angiotensin II synthesis in adrenal and kidney occurs predominantly extracellularly, and is followed by rapid AT1 receptor-mediated endocytosis, thereby leading to high intracellular angiotensin II levels.

Intrarenal angiotensin II: Interstitial and cellular levels and site of production. Background Both local production and angiotensin II subtype 1 (AT 1 ) receptor-mediated uptake from the circulation contribute to the high levels of angiotensin (Ang) II in the kidney. It is largely unknown where Ang II is produced in the kidney and how much of it originates from the circulation. Methods The concentrations of endogenous and 125 I-labeled Ang I and II were measured in renal tissue and in blood from pigs receiving systemic infusions of 125 I-Ang I. Pigs were either untreated or treated with the angiotensin converting enzyme (ACE) inhibitor captopril or the AT 1 receptor antagonist eprosartan. Results 125 I-Ang I was undetectable in renal tissue but the steady-state concentrations of 125 I-Ang II in cortical and medullary tissue were four and two times the concentration in arterial blood plasma, respectively. The tissue concentrations of endogenous Ang II were 100 and 60 times higher than in arterial plasma. Eprosartan reduced 125 I-Ang II accumulation by 90%, but did not lower tissue Ang II. Captopril did not alter either 125 I-Ang II accumulation or tissue Ang II. Conclusions The bulk of Ang II in the kidney is cell-associated. The high tissue/blood concentration ratio of endogenous Ang II may depend on the same mechanism as demonstrated for 125 I-Ang II, that is, AT 1 receptor-mediated binding to cells and endocytosis. If so, the results indicate that most renal AT 1 receptors are exposed to locally generated Ang II rather than Ang II from the circulation. We propose the existence of a low-Ang II vascular system-related interstitial compartment that is separate from tubular fluid, where, according to micropuncture studies, Ang II levels might be high.

A potential role of the sympathetic nervous system in progression of renal failure has received little attention. This study examined whether nonhypotensive doses of moxonidine, an agent that reduces sympathetic activity, affects glomerulosclerosis, urine albumin excretion, and indices of renal handling of norepinephrine (NE) in subtotally nephrectomized (SNX) rats. Sprague Dawley rats were SNX or sham-operated (control). SNX rats were either left untreated or treated with moxonidine in a dose (1.5 mg/kg body wt per d) that did not modify telemetrically monitored 24-h BP. Glomerular and renal morphology were evaluated by quantitative histology, immunohistochemistry, and in situ hybridization. Urine albumin excretion rate was analyzed by enzyme-linked immunosorbent assay, and kidney angiotensin II and NE content were measured using HPLC, (3)H-NE uptake, and release. Body and kidney weight and BP were not significantly different between SNX with or without moxonidine. The glomerulosclerosis index was significantly lower in moxonidine-treated (0.88 +/- 0.09) compared with untreated (1.55 +/- 0.28) SNX rats, as was the index of vascular damage (0.32 +/- 0.14 versus 0.67 +/- 0.16). The number of proliferating cell nuclear antigen-positive glomerular and tubular cells per area was significantly higher in untreated SNX rats than in controls and moxonidine-treated SNX rats. The same was true for urine albumin excretion rate. Renal angiotensin II tissue concentration was not affected by moxonidine. In untreated SNX rats, renal nerve stimulation and exogenous NE induced an increase in isolated kidney perfusion pressure (102 +/- 21 versus 63 +/- 8 mmHg). Renal endogenous NE content was significantly lower in SNX rats than in controls (86 +/- 14 versus 140 +/- 17 pg/mg wet weight). Cortical uptake of [(3)H]-NE was not different, but cortical NE release was significantly higher in SNX rats than in controls. Reduced function of presynaptic inhibitory alpha-adreno-receptors is unlikely because an alpha(2)-adrenoceptor antagonist increased NE release. At subantihypertensive doses, moxonidine ameliorates renal structural and functional damage in SNX animals, possibly through central inhibition of efferent sympathetic nerve traffic. In kidneys of SNX rats, indirect evidence was found for increased activity of a reduced number of nerve fibers.

The existence of a local renin-angiotensin system in the heart is still a controversial issue. This review discusses the evidence, obtained from studies in cardiac cells, in isolated perfused hearts and in intact animals and humans, both under normal and pathological conditions, for local production of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin I and angiotensin II at cardiac tissue sites. In addition, the role of alternative angiotensin-generating enzymes (cathepsin, chymase) and the possibility of (pro)renin uptake from the circulation is evaluated.

Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1 microM), the neurokinin NK1 receptor antagonist L-733060 (0.5 microM), the voltage-sensitive calcium channel blocker ruthenium red (100 microM), the TRPV1 receptor antagonist capsazepine (5 microM), the nitric oxide synthetase inhibitor Nomega-nitro-L-arginine methyl ester HCl (L-NAME; 100 microM), the gap junction blocker 18alpha-glycyrrhetinic acid (10 microM), as well as the RhoA kinase inhibitor Y-27632 (1 microM). Further, we also used the K+ channel inhibitors 4-aminopyridine (1 mM), charybdotoxin (0.5 microM) + apamin (0.1 microM) and iberiotoxin (0.5 microM) + apamin (0.1 microM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) alpha-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK1, NO, vanilloid receptors, voltage-sensitive calcium channels, K+ channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independent mechanism.

To investigate whether renin-angiotensin system blockade might underlie the favorable metabolic effects of the nonselective beta + alpha1-adrenoceptor blocker carvedilol as compared with the selective beta1-adrenoceptor blocker metoprolol.Human coronary microarteries (HCMAs), obtained from 32 heart valve donors, were mounted in myographs.Angiotensin II and the alpha1-adrenoceptor agonist phenylephrine constricted HCMAs to maximally 63 +/- 10 and 46 +/- 15% of the contraction to 100 mmol/l K. Neither carvedilol, metoprolol, the nonselective beta-adrenoceptor antagonist propranolol, nor the alpha1-adrenoceptor antagonist prazosin affected the constrictor response to angiotensin II. alpha1-adrenoreceptors and beta-adrenoceptors are thus not involved in the direct constrictor effects of angiotensin II. When added to the organ bath at a subthreshold concentration, angiotensin II greatly amplified the response to phenylephrine. Both carvedilol and the angiotensin II type 1 (AT1) receptor antagonist irbesartan inhibited this angiotensin II-induced potentiation. Furthermore, carvedilol blocked the angiotensin II-induced amplification of phenylephrine-induced inositol phosphate accumulation in cardiomyocytes.AT1-alpha1-receptor crosstalk, involving inositol phosphates, sensitizes HCMAs to alpha1-adrenoceptor agonists. Our results suggest that, in the presence of an increased sympathetic tone, carvedilol provides AT1 receptor blockade via its alpha1-adrenoceptor blocking effects. This could explain the favorable effects of carvedilol versus metoprolol.

Aldosterone exerts rapid “nongenomic” effects in various nonrenal tissues. Here, we investigated whether such effects occur in the human heart. Trabeculae and coronary arteries obtained from 57 heart valve donors (25 males; 32 females; 17 to 66 years of age) were mounted in organ baths. Aldosterone decreased contractility in atrial and ventricular trabeculae by maximally 34±3% and 15±4%, respectively, within 5 to 15 minutes after its application. The protein kinase C (PKC) inhibitor chelerythrine chloride, but not the mineralocorticoid receptor antagonists spironolactone and eplerenone, blocked this effect. Aldosterone also relaxed trabeculae that were prestimulated with angiotensin II (Ang II), and its negative inotropic effects were mimicked by hydrocortisone (at 10-fold lower potency) but not 17β-estradiol. Aldosterone concentrations required to reduce inotropy were present in failing but not in normal human hearts. Previous exposure of coronary arteries to 1 μmol/L aldosterone or 17β-estradiol (but not hydrocortisone) doubled the maximum contractile response (E max ) to Ang II. ΔE max correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation ( P 1 -adrenoceptor agonist phenylephrine. In conclusion, aldosterone induces a negative inotropic response in human trabeculae (thereby antagonizing the positive inotropic actions of Ang II) and potentiates the vasoconstrictor effect of Ang II in coronary arteries. These effects are specific and involve PKC and ERK 1/2, respectively. Furthermore, they occur in a nongenomic manner, and require pathological aldosterone concentrations.

It has been shown that injection of combinations of anti-aminopeptidase A (APA) monoclonal antibodies (mAb) that inhibit the enzyme activity induces an acute albuminuria in mice. This albuminuria is not dependent on inflammatory cells, complement, or the coagulation system. APA is an important regulator of the renin-angiotensin system because it is involved in the degradation of angiotensin II (Ang II). This study examined the potential role of glomerular Ang II in the induction of albuminuria. The relation among renal Ang II, glomerular APAX enzyme activity, and albuminuria was examined first. Injection of the nephritogenic combinations ASD-3/37 and ASD-37/41 in BALB/c mice induced albuminuria, whereas the non-nephritogenic combination ASD-3/41 had no effect. There was no clear relation between the inhibition of glomerular APA activity and albuminuria, yet it was evident that intrarenal Ang II levels were significantly increased in albuminuric mice and not in nonalbuminuric mice. As a next step, anti-APA mAb were administered to angiotensinogen-deficient mice that do not produce Ang II, and kidney morphology and albuminuria were determined. Angiotensinogen-deficient mice also developed albuminuria upon ASD-37/41 administration. Altogether, these findings clearly demonstrate that Ang II is not required for the induction of albuminuria upon injection of enzyme-inhibiting anti-APA mAb.

Abstract Angiotensin II (Ang II) is internalized by various cell types via receptor-mediated endocytosis. Little is known about the kinetics of this process in the whole animal and about the half-life of intact Ang II after its internalization. We measured the levels of 125 I–Ang II and 125 I–Ang I that were reached in various tissues and blood plasma during infusions of these peptides into the left cardiac ventricle of pigs. Steady-state concentrations of 125 I–Ang II in skeletal muscle, heart, kidney, and adrenal were 8% to 41%, 64% to 150%, 340% to 550%, and 680% to 2100%, respectively, of the 125 I–Ang II concentration in arterial blood plasma (ranges of six experiments). The tissue concentrations of 125 I–Ang I were less than 5% of the arterial plasma concentrations. 125 I–Ang II accumulation seen in heart, kidney, and adrenal was almost completely blocked by a specific Ang II type 1 (AT 1 ) receptor antagonist. Steady-state concentrations of 125 I–Ang II were reached within 30 to 60 minutes in the tissues and within 5 minutes in blood plasma. The in vivo half-life of intact 125 I–Ang II in heart, kidney, and adrenal was approximately 15 minutes, compared with 0.5 minute in the circulation. Thus, Ang II, but not Ang I, from the circulation is accumulated by some tissues, and this is mediated by AT 1 receptors. The time course of this process and the long half-life of the accumulated Ang II support the contention that this Ang II has been internalized after its binding to the AT 1 receptor, so that it is protected from rapid degradation by endothelial peptidases. The results of this study are in agreement with growing evidence of an important physiological role for internalized Ang II.