Walter H. Newman

Evidence for α-1 adrenergic receptor regulation of atriopeptin release from the isolated rat heart

Abstract Atrial myocardium is the source of a recently described peptide hormone termed atriopeptin. Atriopeptin is thought to have a role in the regulation of systemic arterial pressure, fluid balance and plasma electrolyte homeostasis. Isolated rat hearts release atriopeptin into the coronary effluent, and we have found that this release is stimulated by the administration of norepinephrine, a compound with α and β adrenergic properties. Infusion of the pure β-receptor agonist, isoproterenol, failed to stimulate the release; however, the α-1 receptor agonist phenylephrine induced the release in a dose-dependent manner. The stimulation of atriopeptin release by norepinephrine and phenylephrine was inhibited by α-blockade with phentolamine. Administration of BHT-920, a selective α-2 agonist, had no effect on atriopeptin release. We conclude that atriopeptin secretion by the atrial myocyte is stimulated by activation of the α-1 adrenergic receptor. This finding suggests an involvement of the sympathetic nervous system in the physiologic regulation of the secretion of this hormone.

Atrial natriuretic peptide inhibits oxidant-induced increases in endothelial permeability

Chemically and enzymatically generated oxidants alter endothelial cell shape, increase macromolecular permeability across endothelial cell monolayers, and increase lung microvascular permeability. We examined the effect of ANP (atrial natriuretic peptide) on oxidant-induced injuries to bovine aortic endothelial cell monolayers and to isolated, perfused rabbit lungs. Treatment of cultured endothelial monolayers with glucose oxidase (1.4 U/ml) caused changes in cell shape characterized by a retraction of cells and the formation of numerous intercellular gaps. Glucose oxidase treatment also caused a reduction in F-actin stress fibers visualized by rhodamine-phalloidin fluorescence. Pretreatment (5 min) of the endothelial monolayers with ANP (10(-7) M) attenuated the oxidant-induced changes in cell shape and reduction in F-actin staining. In addition, ANP significantly (P less than 0.05) reduced increases in endothelial monolayer permeability to albumin resulting from glucose oxidase treatment. Oxidant-induced injury of isolated, perfused rabbit lungs produced pulmonary edema measured as an increase in lung weight. This increase in weight was significantly (P less than 0.05) inhibited by pretreatment of lungs with ANP (10(-7) M). Collectively, these results suggest that ANP may act to preserve endothelial barrier function and reduce edema formation caused by oxidant injury.

ATRIAL NATRIURETIC PEPTIDE REGULATION OF ENDOTHELIAL PERMEABILITY IS MEDIATED BY CGMP

Previous studies in our laboratory showed that ANP inhibits increases in endothelial monolayer permeability to macromolecules induced by thrombin. In this present study, we investigated the second messenger system involved in the influence of ANP on monolayer permeability. In bovine aortic endothelial cells (BAEC), ANP (100 nM) caused increased cGMP levels which were measurable at 30 sec and maximal at 3 min. Addition of 8-bromo cGMP (1 mM) to BAEC monolayers mimicked the actions of ANP by inhibiting thrombin- mediated increases in permeability to [125I]-labeled bovine serum albumin. Inhibition of increases in permeability by lower concentrations of ANP was enhanced by the cGMP-selective phosphodiesterase inhibitor, M&B 22948 (100 microM). The use of ANP structural analogs which stimulate cGMP production (AP III or BNP) prevented thrombin-induced increases in monolayer permeability, whereas AP-I, which does not increase cGMP levels, was ineffective.

Increased myocardial adenosine release in heart failure

Volume overload congestive heart failure in dogs is associated with a reduced myocardial inotropic responsiveness to the exogenous administration of beta-adrenergic agonists [10, 11]. This same blunted inotropic responsiveness to beta-agonists has now been identified in the failing human myocardium [2]. Volume overload congestive heart failure in dogs is also associated with a reduced resting coronary vascular resistance [7, 12] suggesting the possibility of increased myocardial production of a metabolic vasodilator in the failing heart. Adenosine is a metabolic coronary vasodilator [1] and also has recently been shown to antagonize the inotropic action of beta-adrenergic agonists through a mechanism involving action on the sarcolemmal adenylate cyclase system [4, 13]. Given the findings of blunted inotropic responsiveness of the failing myocardium to beta-adrenergic agonists and reduced coronary vascular resistance in heart failure, we hypothesized that heart failure was associated with elevated myocardial production of adenosine. Accordingly we measured myocardial adenosine release in normal dogs and dogs with volume overload heart failure. Basal levels of myocardial adenosine release were found to be elevated three-fold above normal in dogs with heart failure. It is possible that elevated adenosine release in the failing myocardium contributes both to abnormalities of coronary blood flow and to the blunted inotropic responsiveness of the failing heart to catecholamines.

Ventricular function following acute alcohol administration: A strain-gauge analysis of depressed ventricular dynamics

Abstract Anesthetized open-chest dogs were employed to examine the depressed ventricular dynamics produced by infusions of alcohol and pentobarbital and reversal of this depression with ouabain. Changes in left ventricular contractility, size, and loading conditions (mural force) were continuously monitored with strain gauges. Measurements made from these strain-gauge recordings indicate that the ventricle, depressed with alcohol, operates under increased load. Moreover, compensation for this increased load is through the limited Frank-Starling mechanism. Essentially similar results were obtained with pentobarbital infusions. Infusion of ouabain reversed the depressions, i.e., contractility was increased while mural force and ventricular size decreased. Such findings described the precarious state of ventricular function at moderate blood alcohol levels and may account for the lability of acutely intoxicated patients to hemodynamic stress.

Myocardial Contractile Force as Influenced by Direct Coronary Surgery

Abstract Myocardial contractile force (MCF) measured by direct application of a strain-gauge arch to the left ventricular surface was determined intraoperatively in 11 patients having saphenous vein graft (SVG) bypass of acute or chronic coronary arterial obstruction. The MCF determinations with the grafts open (control), occluded, and released demonstrated a consistent reduction (average 31%) in contractility during graft occlusion and a prompt return to control levels following graft release without alteration in other aspects of ventricular function. Similar changes were not observed during graft occlusion if the arch was applied to nonviable (scarred) myocardium or to areas outside the region of graft perfusion. The SVG augmentation of blood flow to acute or chronically ischemic but viable myocardium enhances MCF or isometric systolic tension, from which coincident improvement in ventricular function should be anticipated.

DIFFERENTIAL EFFECT OF PERTUSSIS TOXIN ON ADENOSINE AND MUSCARINIC INHIBITION OF CYCLIC AMP ACCUMULATION IN CANINE VENTRICULAR MYOCYTES

Cyclic AMP regulation by muscarinic and adenosine receptors was investigated in isolated canine ventricular myocytes. Both the muscarinic receptor agonist, carbachol, and the adenosine receptor agonist, phenylisopropyladenosine, decreased isoproterenol-stimulated cyclic AMP accumulation in a concentration-dependent manner. Carbachol was more potent than phenylisopropyladenosine and had a greater inhibitory effect. At 10(-6) M, carbachol reduced isoproterenol-stimulated cyclic AMP by 73 +/- 5% while 10(-3) M phenylisopropyladenosine was required to decrease cyclic AMP accumulation by 54 +/- 8%. Pretreatment of myocytes with pertussis toxin to inactivate the inhibitory guanine nucleotide binding protein, Gi, completely abolished the effect of phenylisopropyladenosine to reduce cyclic AMP stimulation. In comparison, pertussis toxin treatment blunted the response to carbachol and shifted the dose-effect curve to the right but did not eliminate the inhibitory action of carbachol. In toxin-treated myocytes, 10(-3) M carbachol produced a 26 +/- 6% reduction of isoproterenol-induced cyclic AMP accumulation. This pertussis toxin-insensitive action of carbachol was antagonized by atropine and pirenzepine and was prevented when either of two different phosphodiesterase inhibitors. RO-20-1724 or isobutylmethylxanthine, was included in the incubation medium. The results indicate that adenosine receptor-mediated inhibition of hormone-stimulated cyclic AMP accumulation in ventricular myocytes occurs by a single, Gi-dependent mechanism while muscarinic inhibition appears to involve both Gi-dependent and Gi-independent mechanisms. The Gi-independent mechanism may reflect enhanced phosphodiesterase activity which results from the activation of muscarinic receptors.

A differential effect of ouabain and β-agonists on contractility and lactic acid production in the hypertrophied heart

In response to norepinephrine or isoproterenol, dogs with pressure overload cardiac hypertrophy showed depressed contractility and myocardial lactic acid production when compared to normal dogs. In these dogs with hypertrophy the inotropic response to ouabain remained normal and myocardial lactate production was not induced. This finding is compatible with the idea that the increased oxygen demand induced by beta-agonists exceeds the limited coronary blood flow reserve of the hypertrophied heart leading to myocardial anaerobic metabolism and reduced inotropic responsiveness. Ouabain, on the other hand, did not induce anaerobic metabolism and the inotropic response was not depressed.

Inhibition of endothelial cell clearance of atrial natriuretic peptide by cyclic GMP treatment.

In a previous study, we reported that cyclic GMP (cGMP) selectively down-regulates the clearance receptor (C-receptor) for atrial natriuretic peptide (ANP) in the cultured bovine pulmonary artery endothelial (CPAE) cell line. The present study was undertaken in order to examine the effect of cGMP on the internalization of the ANP-receptor complex in CPAE cells. Maximum binding of [125I]APIII to the cells significantly decreased following the treatment with 1 mM 8-bromo-cGMP for 48 or 72 h. Scatchard analysis of the binding assay data from the treated cells showed a decrease in Bmax (616 to 411 fmol/mg protein) without a significant change in Kd. Removal of cell surface-bound APIII by acetic acid revealed that not only the surface binding, but also the internalization of APIII significantly decreased in 8-bromo-cGMP-treated cells, indicating a decrease in receptor-mediated uptake of ANP into the cells. These results suggest that cGMP regulates the clearance of ANP by vascular endothelial cells.

Blood volume following acute ethyl alcohol ingestion in dogs.

There are conflicting reports regarding blood volume change following alcohol administration. Variations have been reported which are of sufficient magnitude that they could influence any acute circulatory study in which large volumes of alcohol are administered. In these experiments a simple method utilizing a single injection of radioiodinated serum albumin (RISA) was devised. This technique minimizes errors inherent in methods employing multiple injections of RISA. In eight dogs, hematocrit, blood alcohol, and plasma volume were determined at intervals following oral administration of 2 g/kg ethanol (40% v/v in saline) or an equal volume of saline. Blood alcohol peaked at 152 mg/100 ml 3 hr following ingestion. During the 6 hr experimental period there were no significant alterations in hematocrit or plasma volume in either the saline or alcohol treated dogs. These data support the view that blood volume fluctuations are inconsequential in acute cardiovascular experiments performed during 6 hr following ingestion of ethyl alcohol as described above.