Pharmac. Ther. Vol. 48, pp. 417~126, 1990 Printed in Great Britain. All rights reserved

0163-7258/90$0.00+ 0.50 © 1991 PergamonPress plc

Associate Editor: M. J. LEWIS

MECHANISMS OF ATRIAL NATRIURETIC FACTOR-INDUCED VASODILATION RAYMOND J. WINQUIST* a n d THOMAS H. HINTZE'~ *Department of Pharmacology, Boehringer lngelheim Pharmaceuticals Inc., Ridgefield, CT 06877, U.S.A. "~Department of Physiology, New York Medical College Valhalla, N Y 10595, U.S.A. Abstract--ANF can potentially elicit vasorelaxation/n vitro which is typically associated with an elevation in tissue levels of cGMP. Hypotension with vasodilation can be observed upon injection of ANF in vivo, however, infusion of the peptide often results in a decreased blood pressure due to a fall in cardiac output. This apparent discrepancy may reflect some of the distinguishing characteristics of ANF-induced vasorelaxation which include activation of particulate guanylate cyclase, a marked regional vascular selectivity, species differences in the relaxation profile and a variable sensitivity depending on the type and degree of contractile preload.

CONTENTS 1. Introduction 2. Vascular Effects In Vitro 2.1. Vasodilator profile 2.2. Mechanisms of vasorelaxation 2.2.1. Elevation of cyclic GMP 2.2.2. Cyclic GMP-independent mechanisms 2.3. Factors influencing vasorelaxation 2.3.1. Vascular tone 2.3.2. Membrane depolarization 2.3.3. Regional vascular variability 2.3.4. Age 2.3.5. Species differences 3. ANF Receptors 3.1. Receptor subtypes 3.2. Receptors on the endothelium 3.3. Regulation of receptors 4. In Vivo Actions of ANF 4.1. The systemic circulation 4.2. Renal circulation 4.3. Coronary circulation 4.4. Skeletal muscle 4.5. Splanchnic circulation 4.6. Vascular effects of physiologic concentrations of ANF References

1. I N T R O D U C T I O N The discovery of an atrial natriuretic factor (ANF) by de Bold et al. in 1981 started an intensive effort to characterize the pharmacological effects of this substance. Along with causing natriuresis, atrial extracts were shown to possess vasorelaxant activity in several in vitro preparations (Currie et al., 1983; Kleinert et al., 1984; Winquist et al., 1984a). The availability of synthetic material documented that the deduced sequence of A N F was indeed the substance responsible for the vasorelaxation obtained with the atrial extracts (Garcia et al., 1984b; Winquist et al., 1984a). Early work on A N F suggested a unique vasodilator profile in that the peptide acted via a novel mechanism (i.e.

417 418 418 418 418 418 419 419 419 419 420 420 420 420 420 420 421 421 422 422 423 423 423 423

particulate guanylate cyclase, Winquist et al., 1984b) and exhibited a predilection for dilating the renal bed in vivo (e.g. Koike et al., 1984; Hintze et al., 1985). Research efforts subsequent to these early papers have uncovered several controversies including the mechanism of vasorelaxation and if A N F is actually a vasodilator in vivo. This chapter will review both the initial work which characterized the vasorelaxant profile in vitro as well as more recent contributions focusing on whether multiple mechanisms may be responsible for the action of A N F . In addition, the vascular actions of A N F in vivo will be analyzed with attention given to the conditions (e.g. conscious versus anesthetized, bolus versus infusion) which bear importantly on the expression of vasodilator activity. 417

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2. VASCULAR EFFECTS I N V I T R O 2.1. VASODILATORPROFILE

That the relaxation to A N F was a direct action on vascular smooth muscle was indicated by several observations. The relaxation was not affected by antagonists of known autacoids such as antihistamines, beta-adrenergic or cholinergic blockers or indomethacin (Deth et al., 1982; Garcia et al., 1984b). The relaxation response was not influenced by removal of the endothelial lining from the vascular wall (Scivoletto and Carvalho, 1984; Winquist et al., 1984b) and readily occurred in vascular preparations that are relatively devoid of innervation (e.g. rat aorta, Cohen and Schenck, 1985). Moreover, cultured aortic smooth muscle cells were found to contain high affinity receptors for ANF (Hirata et al., 1985). An intriguing development was that ANF was more effective in relaxing contractions induced by hormonal agents (i.e. angiotensin II, norepinephrine) compared to contractions elicited by marked depolarization of the cell membrane by elevated extracellular potassium (Fujii et al., 1986; Garcia et aL, 1984a; Winquist et al., 1984a). This result was opposite to that expected from the organic calcium entry blockers and led some investigators (e.g. Taylor and Meisheri, 1986) to suggest that ANF may be an antagonist of receptor-operated versus potential-dependent calcium channels. The vasodilator profile for ANF was similar to that obtained with the nitrovasodilator class of compounds and therefore led to experiments probing the role of cyclic nucleotides in the vasorelaxant response. 2.2. MECHANISMSOF VASORELAXATION 2.2. I. Elevation o f Cyclic G M P A N F was found to be similar to sodium nitroprusside in elevating cyclic GMP, with no effect on cyclic AMP, coincident with relaxation in rabbit aortic segments (Winquist et al., 1984b). Both the relaxation and elevation of cyclic GMP occurred in the presence or absence of the endothelium. The ability of A N F to elevate cyclic GMP was also found in cultured vascular smooth muscle cells (Hirata et al., 1984) and in nonvascular tissue (e.g. kidney, Hamet et al., 1984). ANF was however distinguished as causing an activation of the particulate isozyme of guanylate cyclase whereas sodium nitroprusside activated the soluble enzyme (Winquist et al., 1984b; Waldman et al., 1984). It has been shown that the relative activities of the particulate and soluble isozymes vary amongst tissues (Tremblay et al., 1985). This may in part explain some of the differences in the vasorelaxant effects found for ANF and the nitrovasodilators discussed below. The relationship between an elevation in cyclic GMP and relaxation is assessed by Lincoln in this series. With regards to an effect of ANF on calcium dynamics, the elevation in cyclic GMP has been linked to blockade of calcium translocation across the plasma membrane (Taylor and Meisheri, 1986), inhibition the release of activator calcium from

intracellular storage sites (Meisheri et al., 1986; Fujii et al., 1986) and activation of a plasma membrane calcium ATPase which would extrude calcium from the inside to the outside of the cell (Fujii et al., 1986). The inhibitory effect of A N F on calcium dynamics has been postulated to occur via inhibition of phosphatidylinositol hydrolysis (Rapoport, 1986). The ANF-mediated increase in cyclic GMP has also been associated with stimulation of Na/K/CI cotransport in vascular smooth muscle (O'Donnell and Owen, 1986). The activation of this antiporter system was dose-dependent and inhibited by prior treatment of tissue with LY 83583, a drug which inhibits the accumulation of intracellular cyclic GMP. However, the significance of this relationship in terms of the vasorelaxant effects of ANF is not clear. An interesting interaction between A N F and amiloride, originally discovered in zona glomerulosa cells (Meloche et al., 1987), has been reported for vascular smooth muscle. Amiloride potentiates the vasorelaxation obtained with A N F in vitro and the ANF-induced hypotension in vivo (Linz et al., 1988). The synergistic effect in vitro was associated with increased levels of cyclic GMP. This potentiating ability of amiloride appears to be due to increasing the coupling of A N F binding to particulate cyclase activation. 2.2.2. Cyclic GMP-Independent Mechanisms Several studies have shown differences in the vasorelaxant profile between ANF and the nitrovasodilators (e.g. Faison et al., 1985) which could be explained on the basis of either an altered distribution of ANF receptors or isozymes of guanylate cyclase. However, the separation of vasorelaxant activity from an ability to elevate cyclic GMP in isolated vascular smooth muscle cells using peptide analogs of A N F (Budzik et aL, 1987) argues for the existence of cyclic GMP-independent mechanisms. Studies examining the inhibition of aidosterone release by ANF in adrenal tissue have often concluded that this effect of A N F is independent of cyclic GMP (e.g. Matsuoka et al., 1985; Elliot and Goodfriend, 1986). Pandey et al. (1987), using adrenal cortex, reported that ANF inhibited the phosphorylation of a 240 kDa plasma membrane protein that was not affected by exogenously added cyclic GMP. Silver et al. (1986) found two high molecular weight proteins (259 and 131 kDa) associated with the particular fraction from rabbit aorta that were phosphorylated with the addition of ANF but not cyclic GMP. Hirata et al. (1988) reported that ANF inhibited the angiotension II-stimulated phosphorylation of the (20 kDa) myosin light chain in cultured rat aortic cells. This effect of A N F was quite modest and an assessment of the effects of cyclic GMP were not included. However, a site of action for A N F beyond the pathways regulating intracellular calcium was suggested by the work of Takuwa and Rasmussen (1987). These authors found that ANF inhibited contractile responses but had variable effects on the level of intracellular calcium in rabbit aorta. Therefore, targets phosphorylated (or prevented from being phosphorylation) by A N F are

ANF-induced vasodilation still unresolved much less whether cyclic GMP has an important role in such effects. A N F has been reported to have a stimulatory effect on basal levels of phosphatidylinositol hydrolysis in cultured vascular smooth muscle cells (Resink et al., 1988). This contrasts with the inhibitory effect of A N F on stimulated (i.e. in the presence of agonist) levels of PI hydrolysis mentioned above. Resink et al. also found that A N F activated phospholipase A2, which, they reasoned, may be a result of the calcium mobilized by the stimulation of PI hydrolysis. These effects of A N F occurred prior to any effect on tissue levels of cyclic GMP. Although the relaxation to A N F is insensitive to indomethacin, the increased release of arachidonic acid may allow for the production of vasodilatory products via lypoxygenase or cytochrome P-450 pathways. Conflicting reports have been published on the effects of ANF on vascular neuromuscular transmission. Negative effects have been published in the blood-perfused rat mesentery (Hezer et al., 1987) and guinea-pig mesenteric and renal arteries (Fujii et al., 1986). However, MacKay and Cheung (1987) found evidence for a presynaptic inhibitory effect in isolated guinea-pig saphenous artery. This inhibitory effect (blockade of synaptic potentials) was not shared by sodium nitroprusside which may reflect a prejunctional site of action (i.e. inhibition of transmitter release) independent of cyclic GMP. Gupta et al. (1989) have found that A N F increased Z2Na uptake in isolated rabbit aortic segments. This action was not mimicked by sodium nitroprusside nor blocked by LY 83583. Although no evidence was found for coupled extrusion of H (i.e. for Na/H exchange), intracellular alkalosis has been associated with relaxation (Denthuluri and Deth, 1989). It should be noted that two receptor subtypes, one coupled to guanylate cyclase and the other uncoupled, possibly serving a clearance function, have been described for several tissues (for review see Winquist and Vlasuk, 1990). It remains possible that the receptor subtype not associated with guanylate cyclase could be linked to another second messenger system associated with vasodilation. 2.3. FACTORSINFLUENCINGVASORELAXATION 2.3.1. Vascular Tone The efficacy of ANF in relaxing agonist-induced contractile responses does depend on the level of preload (Winquist, 1987). Using comparable levels of active force, A N F effectively relaxed contractions elicited by histamine, angiotensin II, norepinephrine and serotonin (Winquist et al., 1984a). Kleinert et al. (1984) found that A N F exerted a predilection for relaxing angiotensin II-versus norepinephrine-induced tone in isolated aortic segments. Faber et al. (1988) demonstrated that A N F completely relaxed aipha~-adrenergic contractions in large arterioles and venules but was inactive against alpha~-adrenergic responses in these vessels. In addition, A N F failed to relax microvessels which had (nonadrenergic) intrinsic tone. Hellegouarch et al. (1988) reported that contractions induced by platelet-activating factor in

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the rat portal vein were refractory to ANF. Therefore, certain agonist-induced responses may be relatively (or completely) insensitive to the relaxant effects of ANF. 2.3.2. Membrane Depolarization Several studies have documented that A N F is more effective in relaxing agonist-induced contractions as compared to contractions elicited by elevated extracellular potassium (for review see Winquist, 1985). This phenomenon may also explain the decrease in efficacy for ANF against contractions resulting from inhibition of the Na/K ATPase or by blockade of outward potassium currents (Winquist et al., 1985) since these responses would be accompanied by cell membrane depolarization. In addition, the phasic activity of the rat portal vein, contractions which are associated with propagated electrical activity, is only partially relaxed by ANF. As discussed above, this profile is near opposite to that obtained with the archtypical calcium entry blockers and thus rendered unlikely blockade of voltage-dependent calcium channels as a mechanism of action. ANF has no discernible effect on membrane potential on isolated vascular preparations (Aalkjaer et al., 1985). The relative decrease in relaxant efficacy in depolarized preparations may explain the differences noted above with various agonists in particular preparations as variable degrees of depolarization no doubt accompany such contractile responses. 2.3.3. Regional Vascular Variability ANF displays marked regional variability in relaxing vascular preparations from animal species (Faison et aL, 1985; Isikawa et al., 1987) and man (Labat et al., 1988). Large conduit arteries are typically more responsive than veins which is surprising in light of the data implicating an effect of ANF on preload versus afterload in vivo (see below). When studied, the variability in relaxation is independent of the manner in which tone is induced. In rabbit vascular preparations, the regional variability was partially correlated with the presence of high affinity receptors on the cell membranes for ANF (Winquist et al., 1985). This finding would be consistent with the variability being manifest as a decrease in maximum relaxation rather than a horizontal shift in the doseresponse curve. However, the correlation was not exact for all preparations such that other factors (e.g. variable degree of depolarization amongst the preparations, distribution of particulate cyclase, differences in the calcium delivery and sequestration pathways) may be operant. The regional variability is also evident in small resistance arteries with renal but not coronary, cerebral nor mesenteric vessels being effectively relaxed by ANF (Aalkjaer et al., 1985; Osol et al., 1986). The vasodilation in the kidney is evident in the afferent but not the efferent arteriolar segments (Loutzenhiser et al., 1988; Ohishi et al., 1988). Interestingly, ANF administration has been associated with efferent arteriolar vasoconstriction, the mechanism of which is unknown (Marin-Grez et al., 1986; Loutzenhiser et al., 1988). This variability

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appears somewhat consistent with in vivo data demonstrating regional differences in the vasodilation obtained with A N F (see below). 2.3.4. Age Vasorelaxant responses were well maintained in several blood vessels taken from Fischer 344 rats up to 27 months of age (Duckies, 1987). As found in other studies referenced above, there was evidence for regional vascular variability in the relaxation response to ANF. 2.3.5. Species Differences Initial studies on atriopeptin I, the 21 amino acid analog of ANF, reported a lack of vasorelaxant efficacy in rabbit aortic strips (Currie et al., 1984). Subsequent studies showed that the peptide could indeed relax this preparation albeit at much higher concentrations than the native peptide (Rapoport et al., 1986). However, if the bioassay was performed in rat aorta, native peptide and atriopeptin I were equi-active. On the other hand, the oxidized product of human sequence A N F (methionine sulfoxide) was very weak in the rat aorta but similar in efficacy to native peptide in the rabbit aorta bioassay (Winquist et al., 1988). Monkey vascular preparations appear to be more sensitive to the vasorelaxant effects of A N F compared to those from rabbit which are more sensitive than canine preparations (Kawai and Ohhashi, 1987). These data show that species differences exist in both the structural requirements of peptide-receptor interaction and ability of the peptide to relax vascular tissue. Wangler et al. (1985) published that A N F caused a coronary vasoconstrictor response when administered in a Langendorff-perfused guinea pig heart assay. This effect was achieved with very high concentrations of A N F (approximately 10 -5 M) and is not observed (coronary vasoconstriction) in dogs (Shapiro et al., 1986; Winquist, 1987). Nonetheless, this may reflect an example of species differences in the coronary vascular responsiveness to ANF.

3. A N F RECEPTORS 3.1. RECEPTOR SUBTYPES

The advent of radioiodinated peptide allowed for characterization of receptor sites on target tissues for ANF. The calculated affinity (low to subnanomolar range) of A N F binding to vascular tissue approximated the effective concentrations eliciting vasorelaxation (Hirata et al., 1985; Napier et al., 1984). However, structure-activity studies utilizing A N F analogs disclosed a discrepancy between binding (all analogs exhibiting similar KdS) and an ability to elevate cyclic GMP (spread over several orders of magnitude) (Leitman and Murad, 1986; Vlasuk et al., 1987). Radioiogic and crosslinking experiments have shown the existence of receptor subtypes in several tissues including vascular (for review see Winquist

and Vlasuk, 1990), which seemingly explained the dissociation noted between the receptor binding and cyclic GMP assays. One receptor species appears linked to particulate guanylate cyclase and shows structural requirements similar to that found in vasorelaxation assays. A second receptor subtype, which is typically in abundance, is not associated with cyclase (Leitman et al., 1986) and exhibits a relative nonselectivity in structural requirements amongst analogs. Thus analogs which are only poorly active in vasorelaxation assays may have a similar relative potency to native peptide in binding assays. This 'uncoupled' receptor may function as a clearance receptor sequestering, and therefore regulating, circulating peptide (Maack et al., 1987). 3.2. RECEPTORS ON THE ENDOTHELIUM

An autoradiographic study in the intact rat demonstrated A N F receptors in the vasculature of kidney, adrenal gland, lung and liver (Bianchi et al., 1985). However, localization of the receptors is complicated by the presence of the high affinity receptors on the vascular endothelium (Schenk et al., 1985; Vlasuk et aL, 1987). The endothelial receptors are apparently not directly associated with vasorelaxation since, as noted above, the relaxation response persists when the endothelium has been removed. Administration of ANF to endothelial cells does result in the elevation of cyclic GMP which implies the existence of the receptor subtype which is coupled to guanylate cyclase (Martin et al., 1988). These receptors may be important for translocating the peptide to a target tissue or in the fluid shifts underlying the hemoconcentration observed with ANF in vivo. In addition, the 'uncoupled' A N F receptors on the endothelial cells may be important in the disposition of circulating peptide. 3.3. REGULATION OF RECEPTORS

Continuous exposure of cultured vascular smooth muscle cells to high concentrations of ANF results in a reduction of A N F receptor number (e.g. Hirata el al., 1987; Roubert et al., 1987). This down-regulation of binding was associated with no change (Hirata et al., 1987) or a concomitant decrease (Roubert et aL, 1987) in the ability of native peptide to elevate the level of cyclic GMP in these cultured cells. Therefore it is not clear whether the 'coupled' receptor subtype can be down-regulated with continuous exposure to ANF. Treatment of cultured cells with phorbol ester does inhibit the ability of ANF to elevate levels of cyclic GMP suggesting that activation of protein kinase C may affect adversely the coupling of the A N F receptor and particulate guanylate cyclase (Nambi et al., 1987; Hirata et al., 1988). However, prolonged exposure of cultured cells with angiotensin II, which might be expected to activate protein kinase C, led to a down-regulation of A N F binding but failed to inhibit the ANF-induced elevation of cyclic GMP (Chabrier et al., 1988). Therefore several factors are no doubt involved in regulating the functional state of the two ANF receptor subtypes.

ANF-induced vasodilation 4. I N

VIVO

ACTIONS OF A N F

The actions of A N F on blood vessels in vivo are dependent upon the type of blood vessel and upon other homeostatic mechanisms which intervene in order to modify the direct actions of ANF. Three blood vessel types: resistance elements in the microcirculation, veins which serve a capacitance function and large muscular arteries, particularly the coronary arteries, all respond to ANF. The homeostatic mechanisms which modify the actions of A N F on blood vessels appear to be the regulation of autonomic outflow by the systemic arterial baroreceptors and cardiopulmonary reflexes, alterations in metabolic demand, for instance in the heart, and in the kidney renal autoregulation. In each organ system, we will point out the direct effects of A N F on each vascular component and what is known about possible modulatory influences. A major controversy concerning the vascular actions of A N F in vivo arose early, based on the apparent disparate mechanisms of vascular regulation following bolus administration, or long term infusion of A N F (Lappe et al., 1985a,b; Shapiro et al., 1986; Patel and Hintze, 1990). This was not based on the method for studying A N F ' s vascular actions but on the physiological function which one envisioned for ANF. For instance, A N F is released transiently during atrial tachyarrythmias in man (Schiffrin et al., 1985) and this is associated with diuresis and natriuresis in some patients. On the other hand, there are measurable plasma levels of A N F at rest and this increases during congestive heart failure (Burnett et al., 1986). The importance of the transient release of A N F versus continuous release is still controversial, however these methods have uncovered two different mechanisms of action of A N F , namely, the direct effect of A N F on vascular smooth muscle and indirect effects which are modulated in part via reflexes and changes in cardiac output. 4.1. THE SYSTEMICCIRCULATION Injection of A N F into dogs and rats invariably results in hypotension (Maack et al., 1984; Hintze et al., 1985; Shapiro et al., 1986), increases in cardiac output (Shapiro et al., 1986) and decreases in calculated total peripheral resistance (Shapiro et al., 1986). Variably these are accompanied by tachycardia in the dog depending upon the initial heart rate (Shapiro et al., 1986), often by tachycardia or no change in heart rate in the rat (Allen and Gellai, 1987; Goetz, 1989) and occasionally by bradycardia in the rat (Thoren et al., 1986). The hypotension, fall in peripheral resistance and increase in cardiac output all appear to be due to the vasodilative actions of A N F on peripheral resistance elements (Seymour et al., 1985; Shapiro et al., 1986). The vasodilation is most pronounced in the renal circulation in the dog and the rat (Oshima et al., 1984; Wakatani et al., 1985; Pegram et al., 1986) and also occurs in skeletal muscle in the anesthetized dog and the rat (Shapiro et al., 1986). The tachycardia appears to result from baroreflex unloading and altered regulation of parasympathetic outflow, a withdrawal of vagal tone to the SA node if there is initially some vagal restraint, and increased sympath-

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etic outflow particularly in the rat and the anesthetized dog (Lappe et al., 1985a; Shapiro et al., 1986; Gellai et al., 1986). This is accompanied by a mild positive inotropic effect which is eliminated by beta adrenergic blockade (Shapiro et al., 1986). Infusion of A N F almost always results in hypotension, reduced cardiac output, an increase in total peripheral resistance and no consistent change in heart rate (Brehaus et al., 1985; Goetz et al., 1986; Lappe et al., 1985b; Pegram et al., 1986; Zimmerman et al., 1987). Patel and Hintze (1990) have recently shown that infusion of Atriopeptin affects the mocardial inotropic state. In these studies calculated cardiac output decreased independent of changes in heart rate or myocardial contractile state. This has led to the speculation that A N F is a vasoconstrictor in support of the data originally published by Maack et al. in the isolated perfused rat kidney (Marin-Grez et al., 1986). In those studies infusion of A N F resulted in a reduction in renal perfusate flow and an increase in G F R , urine flow rate and sodium excretion which were attributed to renal afferent arteriolar dilation and efferent constriction. In vivo, a fall in cardiac output can occur due to peripheral vasoconstriction or reduction in venous return due to an increase in capacitance or reduction in blood volume (Trippodo et al., 1986; Bie et al., 1988). In the anephric rat, infusion of A N F still results in reduced cardiac output, therefore it is not entirely due to an increase in urine flow rate and reduced blood volume (Fluckinger et al., 1986; Aimeida et al., 1987). Studies by Trippodo et al. (1986) have proposed that A N F increases venous resistance to reduce venous return (Yong et al., 1987), whereas others have found that infusion of A N F increases hematocrit, even in the anephric areflexic rat, indicating that A N F may cause fluid to leave the circulation most probably in post capillary venules (Trippodo et al., 1986; Almeida et al., 1987; Holtz et al., 1986). If cardiac output falls then calculated peripheral resistance will increase due either to systemic autoregulation or to baroreflex unloading (Goetz, 1989; Shen et al., 1990b). Venous dilation may be minimal in the dog (Holtz et al., 1986, 1987). There are alternative data to suggest, however, that A N F is a direct vasoconstrictor in the conscious dog. In studies by Shen et al. (1990a), infusion of A N F reduces cardiac output and has little or no effect on systemic arterial pressure resulting in an increase in calculated total peripheral resistance. The increase in resistance is not eliminated by ganglionic or combined adrenergic receptor blockades; is not dependent upon increases in plasma norepinephrine since it is not blocked by alpha adrenergic blockade; and is not dependent upon the release of A D H or activation of the renin angiotensin system since measured levels of these hormones fall. Data from Patel and Hintze (1990) also indicates that infusion of A N F into awake dogs results in no change in arterial pressure, and a fall in cardiac output. The effects of infusion of A N F at 0.5 #g/kg/min for one hour are shown in Fig. 1. One possible explanation for this is venous resistance increases to such a degree that constriction of veins not only reduces venous return, but also increases arteriolar or total peripheral resistance. These results are still a matter of some debate.

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R. J. WINQUISTand T. H. HINTZE

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• combined • unl~ocksd A ganglionic

minutes FIG. l. This figure shows that infusion ofatriopeptin 24 does not reduce total peripheral resistance even in the presence of combined autonomic receptor blockade (propranolol, abropine and pragosin) or ganglionic blockade (hexamethonium). Data such as these have led to the speculation that ANF is a vasoconstrictor in vivo, despite in vitro data suggesting that ANF causes only relaxation of vascular smooth muscle. 4.2. RENALCIRCULATION Perhaps the best studied organ system concerning the effects of A N F is the renal circulation. Early studies by Seymour et al. (1985), Wakitani et al. (1985), Hintze et al. (1985), and Lappe et al. (1985b), showed that injection, or brief infusion of A N F into the rat or the dog, caused only transient increases in renal blood flow. Because these increases were coincident with increases in G F R , it was hypothesized that the increase in renal blood flow was due to afferent arteriolar dilation and may be coincident with efferent constriction (Marin-Grez et al., 1986). This would increase intra-glomerular pressure and increase filtration without having to postulate a change in the filtration constant, kf, of the glomerulus (Huang et aL, 1985). The concept of efferent arteriolar constriction was further supported by the observation that during infusion of A N F into the kidney, renal blood flow fell and G F R increased. In the isolated perfused kidney without preconstriction due to, for instance, the concomitant infusion of norepinephrine, A N F decreased renal perfusate flow. In the presence of some renal tone, infusion of A N F caused vasodilation. These observations were again used to support the contention of selective effects of A N F on the renal afferent and efferent arteriole. Our studies indicated injection or brief infusion (Hintze, 1988) of A N F resulted in renal vasodilation whether using a chronically implanted flow transducer or the radioactive microsphere technique, and that prolonged infusion of A N F resulted in a fall in renal blood flow. The fall in renal blood flow in our studies was eliminated by alpha adrenergic receptor blockade indicating that some efferent arm of the autonomic nervous system was involved. Our studies were supported by the prolonged renal vasodilation in the kidney of the rat after selective renal denervation (Lappe et al., 1985a). Within the kidney, renal blood flow distribution may also change resulting in increased blood flow to the medulla as opposed to the cortex. During the

initial dilation following injection or brief infusion of A N F , early data using radioactive microspheres were interpreted to indicate that A N F caused a selective increase in renal medullary blood flow (Borenstein et al., 1983). Hintze (1988) found that the percent of renal blood flow perfusing the medulla increased in the conscious dog during infusion of ANF, however, the absolute increase in renal blood flow was largest in the cortex. Similar results were found by Hansell and Ulfendahl (1986) using a laser Doppler technique, and reexamination of the initial report by Borenstein et al. (1983) indicates that the absolute increase in renal blood flow to the cortex was larger than to the medulla. Thus instead of selective dilation of blood vessels in the renal medulla, it appears that A N F increases blood flow throughout the kidney, with the largest increase occurring in the cortex and the largest percent change occurring in the medulla. This apparent incongruity stems from the relatively high blood flow in the cortex, approximately 3.5 ml/min/g, as opposed to the medulla, generally less than 1.0 ml/min/g. A 30% increase in blood flow in these two zones of the kidney would cause larger absolute increases in blood flow in the cortex and given that most of the mass of the kidney is cortical, substantial increases in overall renal blood flow (Hintze, 1988). 4.3. CORONARYCIRCULATION Almost universally A N F has been shown to increase coronary blood flow (Shapiro et al., 1986) and large coronary artery diameter (Chu and Cobb et al., 1987) in both anesthetized and awake dogs. Figure 2 shows the effects of increasing doses of A N F given directly into the left circumflex coronary artery in an

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coronary blood flow and fall in calculated mean coronary vascular resistance following intracoronary injections of atriopeptin 24. Although AP24 caused coronary vasadilation at high doses, these were accompanied by diuresis indicating that these levels are sufficient to circulate and affect renal function.

ANF-induced vasodilation anesthetized dog. There were dose dependent increases in coronary blood flow and falls in vascular resistance, however it should be noted that these are extremely large doses of A N F given into a blood flow of approximately 30 ml/min resulting in the coronary arterial plasma levels of in the ng/ml range. Furthermore, these intracoronary doses were sufficient to cause diuresis and naturiesis so that dilution within the whole cardiac output resulted in high enough plasma concentrations to affect the kidney. The most controversial finding in the coronary circulation was the direct vasoconstriction in the guinea pig isolated heart by Wangler et al. (1985). These results have not been confirmed in other species. Alternatively, A N F may reduce myocardial contractile state (Sasaki et al., 1986) and metabolic demand accompanied by reduction in coronary blood flow. 4.4. SKELETALMUSCLE

The data for the skeletal muscle circulation are much less controversial; injection or infusion of A N F has little effect on skeletal muscle blood flow in the conscious dog (Hintze et al., 1985; Shapiro et al., 1986), or in the conscious rat (Lappe et al., 1985b; Gellai et al., 1986). In the anesthetized dog there is some indication that the renal selectivity of A N F disappears and that injection of A N F causes transient decrease in vascular resistance in the gracilis muscle of the dog (Hintze et al., 1985). In the cremasteric microcirculation of the rat, Kaley et al. (1988) found that superfusion of ANF, atriopeptin 23, had no effect on the direct measurement of third order arteriole diameter, although there was some indication that A N F may prevent constriction of these vessels caused by angiotensin II or norepinephrine.

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4.6. VASCULAREFFECTSOF PHYSIOLOGIC CONCENTRATIONSOF A N F Most of the studies described above were conducted using systemic injections or infusions of microgram quantities of A N F into the rat or into the dog. Those studies in which A N F was infused directly into an organ, i.e. the kidney or the heart as discussed above, still infused A N F in microgram quantities. These infusion rates of A N F resulted in plasma concentrations on the order of 5000 pg/ml (Hoegler et al., 1989; Ohanian et al., 1989; Hintze et al., 1988; Shen et al., 1990b; Goetz, 1989). In the conscious dog or monkey, infusion of A N F at 30 ng/kg/min resulted in plasma A N F concentrations of approximately 600 pg/ml. The highest levels of A N F reported in the conscious dog during acute dramatic volume expansion, myocardial infarction, exercise or acute pacing induced tachycardia arc on the order of 300 pg/ml (Hintze et al., 1988, 1989). In man plasma A N F rarely exceeds 100 pg/ml. Even in heart failure plasma A N F is on the order of 500-1000 pg/ml (Burnett et al., 1986). In most of the hemodynamic studies reported above, plasma A N F was given in pharmacologic concentrations, that is above 1000 pg/ml. At these doses A N F caused only modest vasodilation in any vascular bed, and consistent vasodilation only in the renal circulation.

REFERENCES AALIOAER, C., MULVANY, M. J. and NVaORG, N. C. B.

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Atrial ~atriuretic factor has no significant effect on blood flow in the superior mesenteric artery of the conscious dog (Hintze et al., 1985). Using radioactive microspheres at a point in time where injection of A N F increased renal blood flow, blood flow in the stomach, the small and large intestines, the pancreas and the liver did not change (Shapiro et al., 1986). Blood flow in isolated mesenteric arterioles of the cat did not change. There is some data which indicates that the weight of the isolated intestine increases during the infusion of A N F which has been used to support the conclusion that A N F causes an increase in filtration (Osol et al., 1986). Whether this is due to a change in permeability of the capillaries in the gut or to a change in the hydrostatic pressure across the mesenteric capillary is not clear. Shapiro et al. (1986) found that injection of A N F resulted in an increase in blood flow to the spleen which was concurrent with an increase in splenic dimensions. The function of relaxation of the spleen is not known, however the spleen is an important blood reservoir in the dog, and this observation may support the function of A N F as a hormone involved in the regulation of systemic blood volume. JPT 4S/3--J

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