Duncan J. Campbell

Sympathetic Augmentation in Hypertension: Role of Nerve Firing, Norepinephrine Reuptake, and Angiotensin Neuromodulation

Abstract—There is growing evidence that essential hypertension is commonly neurogenic and is initiated and sustained by sympathetic nervous system overactivity. Potential mechanisms include increased central sympathetic outflow, altered norepinephrine (NE) neuronal reuptake, diminished arterial baroreflex dampening of sympathetic nerve traffic, and sympathetic neuromodulation by angiotensin II. To address this issue, we used microneurography and radiotracer dilution methodology to measure regional sympathetic activity in 22 hypertensive patients and 11 normotensive control subjects. The NE transport inhibitor desipramine was infused to directly assess the potential role of impaired neuronal NE reuptake. To evaluate possible angiotensin sympathetic neuromodulation, the relation of arterial and coronary sinus plasma concentrations of angiotensin II to sympathetic activity was investigated. Hypertensive patients displayed increased muscle sympathetic nerve activity and elevated total systemic, cardiac, and renal NE spillover. Cardiac neuronal NE reuptake was decreased in hypertensive subjects. In response to desipramine, both the reduction of fractional transcardiac 3[H]NE extraction and the increase in cardiac NE spillover were less pronounced in hypertensive patients. DNA sequencing analysis of the NE transporter gene revealed no mutations that could account for reduced transporter activity. Arterial baroreflex control of sympathetic nerve traffic was not diminished in hypertensive subjects. Angiotensin II plasma concentrations were similar in both groups and were not related to indexes of sympathetic activation. Increased rates of sympathetic nerve firing and reduced neuronal NE reuptake both contribute to sympathetic activation in hypertension, whereas a role for dampened arterial baroreflex restraint on sympathetic nerve traffic and a peripheral neuromodulating influence of angiotensin II appear to be excluded.

AMPK β Subunit Targets Metabolic Stress Sensing to Glycogen

Abstract AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients [1], as well as hormones, including adiponectin and leptin [2, 3]. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK [4]. Mammalian AMPK is an αβγ heterotrimer with multiple isoforms of each subunit comprising α1, α2, β1, β2, γ1, γ2, and γ3, which have varying tissue and subcellular expression [5, 6]. Mutations in the AMPK γ subunit cause glycogen storage disease in humans [7], but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK β subunit contains a functional glycogen binding domain (β-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished β-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK β1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.

Differential regulation of angiotensin peptide levels in plasma and kidney of the rat.

We compared the effects of the converting enzyme inhibitor perindopril on components of the renin-angiotensin system in plasma and kidney of male Sprague-Dawley rats administered perindopril in their drinking water at two doses (1.4 and 4.2 mg/kg) over 7 days. Eight angiotensin peptides were measured in plasma and kidney: angiotensin-(l–7), angiotensin II, angiotensin-(l–9), angiotensin I, angiotensin-(2–7), angiotensin III, angiotensin-(2–9), and angiotensin-(2–10). In addition, angiotensin converting enzyme activity, renin, and angiotensinogen were measured in plasma, and renin, angiotensinogen, and their respective messenger RNAs were measured in kidney; angiotensinogen messenger RNA was also measured in liver. In plasma, the highest dose of perindopril reduced angiotensin converting enzyme activity to 11% of control, increased renin 200-fold, reduced angiotensinogen to 11% of control, increased angiotensin-(l–7), angiotensin I, angiotensin-(2–7), and angiotensin-(2-10) levels 25-, 9-, 10-, and 13-fold, respectively; angiotensin II levels were not significantly different from control. By contrast, for the kidney, angiotensin-(l–7), angiotensin I, angiotensin-(2–7), and angiotensin- (2–10) levels did not increase; angiotensin II levels fell to 14% of control, and angiotensinogen fell to 12% of control. Kidney renin messenger RNA levels increased 12-fold, but renal renin content and angiotensinogen messenger RNA levels in kidney and liver were not influenced by perindopril treatment. These results demonstrate a differential regulation of angiotensin peptides in plasma and kidney and provide direct support for the proposal that the cardiovascular effects of converting enzyme inhibitors depend on modulation of tissue angiotensin systems. Moreover, the failure of kidney angiotensin I levels to increase with perindopril treatment, taken together with the fall in kidney angiotensinogen levels, suggests that angiotensinogen may be a major rate-limiting determinant of angiotensin peptide levels in the kidney.

Mice with Cardiac-Restricted Angiotensin-Converting Enzyme (ACE) Have Atrial Enlargement, Cardiac Arrhythmia, and Sudden Death

To investigate the local effects of angiotensin II on the heart, we created a mouse model with 100-fold normal cardiac angiotensin-converting enzyme (ACE), but no ACE expression in kidney or vascular endothelium. This was achieved by placing the endogenous ACE gene under the control of the alpha-myosin heavy chain promoter using targeted homologous recombination. These mice, called ACE 8/8, have cardiac angiotensin II levels that are 4.3-fold those of wild-type mice. Despite near normal blood pressure and a normal renal function, ACE 8/8 mice have a high incidence of sudden death. Both histological analysis and in vivo catheterization of the heart showed normal ventricular size and function. In contrast, both the left and right atria were three times normal size. ECG analysis showed atrial fibrillation and cardiac block. In conclusion, increased local production of angiotensin II in the heart is not sufficient to induce ventricular hypertrophy or fibrosis. Instead, it leads to atrial morphological changes, cardiac arrhythmia, and sudden death.

Nephrectomy, converting enzyme inhibition, and angiotensin peptides.

To determine the contribution of kidney-derived renin and angiotensin converting enzyme to circulating and tissue levels of angiotensin peptides, we measured angiotensin (Ang)-(1-7), Ang II, Ang-(1-9), and Ang I in plasma, kidney, lung, heart, aorta, brown adipose tissue, adrenal, pituitary, and brain of five groups of male Sprague-Dawley rats: control rats, rats given the converting enzyme inhibitor ramipril (10 mg/kg), rats nephrectomized 24 hours, rats nephrectomized 48 hours, and rats nephrectomized 48 hours and given ramipril. Plasma and tissues, apart from adrenal, showed a 63% to 98% reduction in Ang II, the ratio of Ang II to Ang I, or both after ramipril administration, indicating a major role for converting enzyme in Ang II formation. Nephrectomy caused a more than 95% decrease in plasma renin levels and a fourfold to eightfold increase in plasma angiotensinogen levels. Apart from plasma and brain, tissues showed a 59% to 78% decrease in Ang II levels after nephrectomy, indicating a major role for kidney-derived renin in Ang II formation. The persistence of Ang II in plasma and tissues of anephric rats indicates that Ang II may be formed by a process independent of kidney-derived renin; this process may be amplified by the increased plasma angiotensinogen levels that accompany nephrectomy. For lung, adrenal, and aorta, Ang II levels showed a further decrease when nephrectomized rats were given ramipril. However, for plasma and the other tissues, ramipril produced little or no decrease in Ang II levels of anephric rats, suggesting that Ang II may be formed by a pathway independent of converting enzyme. Such a pathway may involve the direct formation of Ang II from angiotensinogen by a non-renin-like enzyme.

Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation

Extracellular nucleotides play an important role in thrombosis and inflammation, triggering a range of effects such as platelet activation and recruitment, endothelial cell activation, and vasoconstriction. CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase), converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inflammatory mediator adenosine. Deletion of CD39 renders mice exquisitely sensitive to vascular injury, and CD39-null cardiac xenografts show reduced survival. Conversely, upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inflammation. In this study we examined the consequences of transgenic expression of human CD39 (hCD39) in mice. Importantly, these mice displayed no overt spontaneous bleeding tendency under normal circumstances. The hCD39 transgenic mice did, however, exhibit impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism. Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived longer in a mouse cardiac transplant model of vascular rejection. These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates.

Losartan increases bradykinin levels in hypertensive humans.

Background—Studies in animals and humans indicate a role for kinins in the actions of angiotensin type 1 (AT1) receptor blockers. However, the effect of these compounds on kinin levels in humans is unknown. Methods and Results—We measured angiotensin (Ang), bradykinin (BK), and kallidin peptides in subjects with essential hypertension administered placebo, losartan (50 mg OD), and eprosartan (600 mg OD) in randomized order in a double-blind, 3-period, 3-treatment, crossover trial. Peptides were measured in arterial blood using high-performance liquid chromatography–based radioimmunoassays. Losartan increased blood levels of BK-(1–9) and hydroxylated BK-(1–9) by ≈2-fold and reduced the BK-(1–7)/BK-(1–9) ratio by 55%. There was a trend for eprosartan to produce similar changes in bradykinin levels. There were no changes in blood kallidin levels. Both losartan and eprosartan increased plasma levels of Ang I, Ang II, and Ang-(2–8), and eprosartan increased Ang-(3–8) levels. Ang-(1–7) and Ang-(1–9) levels were unchanged. There was an associated 30% to 35% reduction in Ang II/Ang I ratio and 63% to 69% reduction in Ang-(1–7)/Ang I ratio. Plasma ACE activity was unchanged. Conclusions—Losartan increases bradykinin levels. The reductions in BK-(1–7)/BK-(1–9), Ang II/Ang I, and Ang-(1–7)/Ang I ratios suggest that the increased bradykinin levels were the result of reduced metabolism by ACE and neutral endopeptidase. Increased bradykinin levels may represent a class effect of AT1 receptor blockers that contributes to their therapeutic actions and may also contribute to the angioedema that may accompany this therapy.

Bradykinin peptides in kidney, blood, and other tissues of the rat.

The bradykinin peptide system is a tissue-based system with potent cardiovascular and renal effects. To investigate the regulation of this system, we developed a highly sensitive amino terminal-directed radioimmunoassay that, with high performance liquid chromatography, enables the measurement of bradykinin-(1-7), bradykinin-(1-8), and bradykinin-(1-9). Together with a carboxy terminal-directed radioimmunoassay, we characterized bradykinin peptides in rat kidney and blood. The predominant bradykinin peptides in kidney were bradykinin-(1-9) (approximately 100 fmol/g wet weight of tissue) and bradykinin-(1-7) (approximately 70 fmol/g), with low levels of bradykinin-(1-8) (approximately 8 fmol/g) and bradykinin-(4-9) (approximately 12 fmol/g) detectable; bradykinin-(2-9) and bradykinin-(3-9) were below the limits of detection. In blood, the levels of bradykinin-(1-9) were very low (approximately 2 fmol/ml), and other bradykinin peptides were below the limits of detection. Ile,Ser-bradykinin and Met,Ile,Ser-bradykinin were below the limits of detection in both kidney and blood, indicating that T-kininogen makes no detectable contribution to renal or circulating bradykinin peptides. Administration of the angiotensin converting enzyme inhibitor perindopril was associated with an approximate twofold increase in renal levels of bradykinin-(1-8) and bradykinin-(1-9) and a decrease in the bradykinin-(1-7)/bradykinin-(1-9) ratio. The amino terminal-directed radioimmunoassay was also applied to heart, aorta, brown adipose tissue, adrenal lung, and brain. For these tissues, bradykinin-(1-7) and bradykinin-(1-9) were of similar abundance (16-340 fmol/g), with lower levels of bradykinin-(1-8). These studies demonstrate that tissue levels of bradykinin peptides are much higher than circulating levels, consistent with their formation at a local tissue site. Of peptides derived from K-kininogen, bradykinin-(1-9) is the predominant bioactive peptide in all tissues, and a major pathway of bradykinin-(1-9) metabolism involves the formation of bradykinin-(1-7). In kidney, angiotensin converting enzyme plays an important role in bradykinin-(1-9) metabolism, and increased bradykinin-(1-9) and bradykinin-(1-8) levels may mediate in part the renal effects of converting enzyme inhibition.

Effects of losartan on angiotensin and bradykinin peptides and angiotensin-converting enzyme

Antagonists of the type 1 (AT1) angiotensin II (Ang II) receptor increase renin secretion and plasma Ang II levels, and the increased Ang II levels may counteract the effects of the antagonist. Moreover, other investigators have suggested that the reactive increase in Ang II levels may increase bradykinin (BK) levels through stimulation of the type 2 Ang II receptor (AT2). We investigated the acute effects of the AT1 receptor antagonist losartan (intraarterial injection of 10 mg/kg every 12 h) in male Sprague Dawley rats by measuring circulating angiotensin and BK peptides at 6, 12, and 24 h. Whereas acute losartan administration increased blood angiotensin levels four- to sixfold, blood BK levels were unchanged. We also investigated the effects of losartan administered for 8 days (10 mg/kg every 12 hours, by intraperitoneal injection) on circulating and tissue levels of angiotensin and BK peptides, and angiotensin-converting enzyme (ACE). Losartan increased plasma renin levels 100-fold; plasma angiotensinogen levels decreased to 24% of control; and plasma aldosterone levels were unchanged. Ang II levels in plasma, adrenal, lung, heart, and aorta were increased 25-, 8-, 3.5-, 2.4-, and 14-fold, respectively, by losartan administration. By contrast, kidney Ang II levels decreased to 71% of control, accompanied by a decrease in kidney levels of BK-(1–7) and BK-(1–9). No other tissue showed a change in BK peptide levels, except for a reduction in blood levels of BK-(1–8) to 43% of control. Plasma ACE increased by 13–50%, but tissue ACE levels were unchanged. These data demonstrate that losartan has tissue-specific effects on endogenous levels of angiotensin and BK peptides and indicate that increased BK levels do not contribute to the actions of losartan. The absence of a reactive increase in endogenous kidney levels of Ang II indicates that this tissue is likely to be the most sensitive to AT1 receptor antagonism.

Synergistic effects of ACE inhibition and Ang II antagonism on blood pressure, cardiac weight, and renin in spontaneously hypertensive rats.

BACKGROUND Blockade of type 1 angiotensin (Ang) II receptors combined with ACE inhibition may amplify the efficacy of the renin-angiotensin system blockade because ACE inhibitors do not completely and permanently suppress Ang II production. METHODS AND RESULTS Enalapril or losartan (1, 3, 10, and 30 mg/kg) or their combination was administered for 2 to 4 weeks to spontaneously hypertensive rats. The combination of low doses of each agent induced greater reductions in blood pressure (BP) and left ventricular weight/body weight (LVW/ BW) ratio than monotherapy with the same or higher doses. When approximately equipotent regimens of enalapril, losartan, and their combination, as judged by BP fall, were compared, there were similar increases in plasma and renal renin and in plasma Ang-(1-7) and Ang I and similar reductions in plasma angiotensinogen. Enalapril alone reduced plasma Ang II levels, and losartan alone increased Ang II levels. The combination of enalapril with losartan prevented or reduced the increase in Ang II levels observed with losartan alone. CONCLUSIONS These findings show that the synergistic interaction between the effects of low doses of enalapril and losartan on BP and LVW/BW ratio is due to more effective inhibition of the renin-angiotensin system by their combination than by either agent alone. When both drugs are given together, the ACE inhibitor-induced fall in plasma Ang II results in modulation of the Ang II antagonist-induced reactive rise in Ang II, thereby lowering the plasma Ang II concentration, which competes with the antagonist for the Ang II receptors.