Robert L. Rodgers

Effects of Doxazosin on Vascular Collagen Synthesis, Arterial Pressure and Serum Lipids in the Spontaneously Hypertensive Rat

Hypertension in various experimental models, including spontaneously hypertensive rats (SHR), is associated with elevated rates of vascular collagen synthesis. The sympathetic nervous system is an important factor in the etiology of hypertension in SHR. The primary purpose of this study was to determine the effects of the α1adrener-gic receptor antagonist doxazosin on aortic collagen synthesis and on systolic arterial pressure in SHR. Doxazosin was administered either short-term (20 or 200 mg/kg/day by gavage over 5 days) or long-term (0.02 or 0.20 g/L in the drinking water over 8 weeks). Rates of collagen synthesis were determined by incubating aortic segments with 14C-proline in vitro and then measuring either the formation of 14C-hydroxyproline by means of high-performance liquid chromatography, or the amount of radioactivity liberated by collagenase digestion. Systolic arterial pressure was monitored with the standard tail-cuff technique. Both doses of doxazosin depressed aortic collagen synthesis at 8 weeks of treatment, but neither dose had any effect at 4 weeks. In the short-term study only the higher acute dose of doxazosin significantly reduced aortic collagen synthesis; the lower dose had no effect. In the short-term study doxazosin reduced systolic arterial pressure, with a maximum effect at 1–2 days. Tolerance to the depressor effect developed over the remaining 3–4 days, especially with the higher dose. In the 8-week study, the lower doxazosin dose had no effect on systolic arterial pressure, and the higher dose exerted a biphasic effect, moderately but significantly reducing systolic arterial pressure at 1 and 8 weeks of treatment. Assays of serum doxazosin levels suggested that the tolerance to the depressor effect was partially, but not completely, pharmacokinetic. The higher dose of doxazosin significantly reduced serum triglycerides, but none of the other treatment regimens affected serum lipid profiles (very-low-density, low-density or high-density lipoproteins or total cholesterol). The results suggest that the inhibition of vascular collagen synthesis by doxazosin in SHR may be to some extent independent of reductions in arterial pressure.

Ventricular relaxation of diabetic spontaneously hypertensive rat.

Diabetes, and possibly the hypothyroidism that attends diabetes, impairs mechanical relaxation of ventricular muscle, in part by depressing the rate of Ca2+ uptake by sarcoplasmic reticulum. Left ventricular hypertrophy exacerbates the adverse effects of diabetes on cardiac performance, but its effects on relaxation variables have not been well characterized. We examined the impact of streptozotocin-induced diabetes (8 weeks) on ventricular pressure load-dependent relaxation and sarcoplasmic reticular calcium uptake of hearts from spontaneously hypertensive rats and Wistar-Kyoto rats. Subsets of diabetic hypertensive rats were treated with either insulin (10 units/kg/day) or triiodothyronine (8-10 micrograms/kg/day). Diabetes impaired load-dependent relaxation and depressed sarcoplasmic reticular calcium uptake only in spontaneously hypertensive rat hearts. Either insulin or triiodothyronine treatment prevented the diabetes-induced depressions of both mechanical and biochemical indexes of relaxation. The results suggest that 1) hypertrophic ventricles of spontaneously hypertensive rats are more susceptible to the detrimental effects of diabetes on relaxation indexes than are the nonhypertrophic Wistar-Kyoto rat ventricles, and 2) the hypothyroidism that attends diabetes may contribute to the impaired relaxation of diabetic spontaneously hypertensive rat left ventricle.

Insulin-like effects of a physiologic concentration of carnitine on cardiac metabolism

Pharmacologic (millimolar) levels of carnitine have been reported to increase myocardial glucose oxidation, but whether physiologically relevant concentrations of carnitine affect cardiac metabolism is not known. We employed the isolated, perfused rat heart to compare the effects of physiologic levels of carnitine (50 μM) and insulin (75 mU/l [0.5 nM]) on the following metabolic processes: (1) glycolysis (release of 3H2O from 5-3H-glucose); (2) oxidation of glucose and pyruvate (production of 14CO2 from U-14C-glucose, 1-14C-glucose, 3,4-14C-glucose, 1-14C-pyruvate, and 2-14C-pyruvate); and (3) oxidation of palmitate (release of 3H2O from 9,10-3H-palmitate). We found that addition of carnitine (50 μM) to a perfusate containing both glucose (10 mM) and palmitate (0.5 mM) stimulated glycolytic flux by 20%, nearly doubled the rate of glucose oxidation, and inhibited palmitate oxidation by 20%. These actions of carnitine were uniformly similar to those of insulin. When carnitine and insulin were administered together, their effects on the oxidation of glucose and palmitate, but not on glycolysis, were additive. When pyruvate (1 mM) was substituted for glucose, neither carnitine nor insulin influenced the rate of oxidation of pyruvate or palmitate. In combination, however, carnitine and insulin sharply suppressed pyruvate oxidation (75%) and doubled the rate of palmitate oxidation. None of the responses to carnitine or insulin was affected by varying the isotopic labeling of glucose or pyruvate. The results show that carnitine, at normal blood levels, exerts insulin-like effects on myocardial fuel utilization. They also suggest that plasma carnitine in vivo may interact with insulin both additively and permissively on the metabolism of carbohydrates and fatty acids

Cardiac Glucose and Fatty Acid Oxidation in the Streptozotocin-Induced Diabetic Spontaneously Hypertensive Rat

Hypertension intensifies the cardiac dysfunction of diabetes. We investigated the possible role of altered exogenous fuel oxidation in this phenomenon. Diabetes was induced by streptozotocin in spontaneously hypertensive rats and normotensive Sprague-Dawley rats. Two weeks later, mechanical performance and the oxidation of glucose and palmitate were quantified in working hearts ex vivo at intermediate and high workloads. The results showed that the nondiabetic spontaneously hypertensive rat hearts, compared with those of the normotensive controls, oxidized glucose at a higher rate but oxidized palmitate at a much lower rate, as reported previously. The effects of diabetes in the hypertensive rats, compared with its effects in the normotensive strain, were characterized by (1) a more pronounced decrease in heart performance, (2) either a similar or a less marked reduction in the rate of glucose oxidation, depending on the workload, and (3) a relatively greater increase in palmitate oxidation, particularly at the higher workload. These findings suggest that the exaggerated stimulation of fatty acid oxidation by diabetes in the hypertrophic left ventricle may be a more important contributor to the premature mechanical dysfunction than the inhibition of glucose oxidation. Possible mechanisms include antagonism of energetically favorable shifts in fuel oxidation or inhibition of accelerated membrane lipid biosynthesis in left ventricular hypertrophy.

Insulin-like stimulation of cardiac fuel metabolism by physiological levels of glucagon: involvement of PI3K but not cAMP

At concentrations around 10(-9) M or higher, glucagon increases cardiac contractility by activating adenylate cyclase/cyclic adenosine monophosphate (AC/cAMP). However, blood levels in vivo, in rats or humans, rarely exceed 10(-10) M. We investigated whether physiological concentrations of glucagon, not sufficient to increase contractility or ventricular cAMP levels, can influence fuel metabolism in perfused working rat hearts. Two distinct glucagon dose-response curves emerged. One was an expected increase in left ventricular pressure (LVP) occurring between 10(-9.5) and 10(-8) M. The elevations in both LVP and ventricular cAMP levels produced by the maximal concentration (10(-8) M) were blocked by the AC inhibitor NKY80 (20 microM). The other curve, generated at much lower glucagon concentrations and overlapping normal blood levels (10(-11) to 10(-10) M), consisted of a dose-dependent and marked stimulation of glycolysis with no change in LVP. In addition to stimulating glycolysis, glucagon (10(-10) M) also increased glucose oxidation and suppressed palmitate oxidation, mimicking known effects of insulin, without altering ventricular cAMP levels. Elevations in glycolytic flux produced by either glucagon (10(-10) M) or insulin (4 x 10(-10) M) were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (10 microM) but not significantly affected by NKY80. Glucagon also, like insulin, enhanced the phosphorylation of Akt/PKB, a downstream target of PI3K, and these effects were also abolished by LY-294002. The results are consistent with the hypothesis that physiological levels of glucagon produce insulin-like increases in cardiac glucose utilization in vivo through activation of PI3K and not AC/cAMP.

Depressor effect of diabetes in spontaneously hypertensive rat: Role of vascular reactivity and prolyl hydroxylase and lysyl oxidase activities

Streptozotocin (STZ)-induced diabetes (8 weeks) produced a marked depressor effect in the spontaneously hypertensive rat (SHR), confirming earlier studies, but had no effect on arterial pressure of normotensive controls (WKY). We investigated the phenomenon further by examining the effects of diabetes on the activities of aortic prolyl hydroxylase (PH) and lysyl oxidase (LO), marker enzymes for collagen biosynthesis, and on the reactivity of isolated mesenteric arteries to vasoactive agents. PH and LO activities of nondiabetic SHR were greater than those of the WKY controls. Diabetes markedly reduced PH and LO activities of SHR aortae, but had no significant effect on PH and LO activities of the WKY strain. The effects of diabetes on vascular collagen biosynthetic enzymes of SHR were not associated with reductions in mesenteric arterial responsiveness or sensitivity to norepinephrine, methoxamine, serotonin or KC1. These results suggest that the depressor effect of diabetes in SHR is associated with a reduction in vascular collagen biosynthesis but not a reduction in vascular reactivity.

Positive inotropic and toxic effects of brevetoxin-B on rat and guinea pig heart☆

Brevetoxin-B (GbTX-B), a cyclic polyether purified from the marine dinoflagellate Gymnodinium breve, produced positive inotropic and arrhythmogenic effects on isolated rat and guinea pig cardiac preparations at concentrations between 1.25 X 10(-8) and 1.87 X 10(-7) M. The toxin (10(-7) M) transiently increased left ventricular +dP/dt, hydraulic work, and oxygen consumption of paced working rat hearts, then reduced these variables during continuous exposure. Brevetoxin-B exerted a much smaller positive inotropic effect on working guinea pig hearts, but produced a marked and sustained inotropic effect on guinea pig left atria. The toxin also produced arrhythmias in rat and guinea pig hearts, characterized by ventricular tachycardia and A-V blockade. Sympatholytic procedures (beta blockade or reserpine pretreatment) partially blocked the positive inotropic effects, and eliminated the ventricular tachycardia, but not the A-V blockade. Tetrodotoxin markedly inhibited the positive inotropic effect of GbTX-B. Brevetoxin-B did not inhibit guinea pig cardiac Na,K-ATPase activities. The results show that GbTX-B is a potent cardiotoxin and suggest that GbTX-B exerts positive inotropic and arrhythmogenic effects by increasing sarcolemmal sodium permeability, and by releasing catecholamines from sympathetic nerve endings.

Mechanical function, glycolysis, and ultrastructure of perfused working mouse hearts following thoracic aortic constriction.

BACKGROUND Glycolytic flux in the mouse heart during the progression of left ventricular hypertrophy (LVH) and mechanical dysfunction has not been described. METHODS The main objectives of this study were to characterize the effects of thoracic aortic banding, of 3- and 6-week duration, on: (1) left ventricular (LV) systolic and diastolic function of perfused working hearts quantified by analysis of pressure-volume loops; (2) glycolytic flux in working hearts expressed as the rate of conversion of (3)H-glucose to (3)H(2)O, and (3) ultrastructure of LV biopsies assessed by quantitative and qualitative analysis of light and electron micrographs. RESULTS Results revealed that (1) indexes of systolic function, including LV end-systolic pressure, cardiac output, and rate of LV pressure development and decline, were depressed to similar degrees at 3 and 6 weeks post-banding; (2) diastolic dysfunction, represented by elevated LV end-diastolic pressure and volume, was more severe at 6 than at 3 weeks, consistent with a transition to failure; (3) a progressive decline in glycolytic flux that was roughly half the control rate by 6 weeks post-banding; and (4) structural derangements, manifested by increases in interstitial collagen content and myocyte Z-band disruption, that were more marked at 3 weeks than at 6 weeks. CONCLUSION The results are consistent with the view that myocyte damage, fibrosis, and suppressed glycolytic flux represent maladaptive structural and metabolic remodeling that contribute to the development of failure in high pressure load-induced LVH in the mouse.

STZ-induced diabetes in SHR and renovascular hypertensive rats : dissociation between changes in arterial pressure and vascular collagen synthesis

Streptozotocin (STZ)-induced diabetes depresses the rate of vascular collagen synthesis in the spontaneously hypertensive rat (SHR), but it also reduces arterial pressure (SAP) in this strain. We investigated this phenomenon further by comparing the SHR with the renovascular hypertensive (RVH) rat, because diabetes does not affect SAP in the latter model of hypertension. Renovascular hypertension was induced by clipping the left renal artery of Wistar-Kyoto (WKY) rats; sham-operated WKY were included as normotensive controls. Collagen synthesis of arterial tissue in vitro was quantified as prolyl hydroxylase activity and the rate of radioactive proline incorporation into collagen. Arterial collagen synthesis of nondiabetic SHR and RVH animals was elevated compared to that of the nonhypertensive WKY controls. STZ-induced diabetes (8 weeks) reduced SAP of SHR, but had no effect on SAP of either RVH or normotensive WKY rats. However, diabetes significantly depressed vascular collagen synthesis of both SHR and RVH rats, and, less consistently, of the WKY. The results strongly suggest that STZ-induced diabetes in SHR impairs arterial collagen synthesis independent of associated changes in arterial pressure.

The isolated perfused working mouse heart system

Study of cardioactive agents in vivo is complicated by neurohormonal influences and variations in loading conditions. The working mouse heart perfusion system allows for the ex vivo direct measurement of cardiac metabolism and function, and responses to drugs and hormones, under defined conditions. Myocardial pressure and volume measurements are obtained using a micro-tip catheter inserted into the left ventricular chamber. Direct and derived functional data include heart rate (HR), cardiac output (CO), end systolic pressure (ESP), end diastolic pressure (EDP), end systolic volume (ESV), end diastolic volume (EDV), stroke volume (SV), and ejection fraction (EF). Metabolic data include rates of glycolysis, glucose oxidation, and fatty acid oxidation. The mouse serves as an ideal model for evaluation of the pharmacology and toxicity of novel cardioactive therapeutics due to its rapid reproduction, low cost, and relevance to human physiology.