Growth-regulatory

properties

RICHARD

G. APPEL

Department

of Medicine,

Bowman

of atria1 natriuretic

Gray School of Medicine,

Winston-Salem,

factor North

Carolina

27157-1053

Appel, Richard G. Growth-regulatory properties of atria1 natriuretic factor. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F911-F918, 1992.-Recent evidence supports the notion that atria1 natriuretic factor (ANF) has growth-regulatory properties. In the adrenal gland, ANF inhibits growth specifically in the zona glomerulosa. In the kidney, ANF causes an antimitogenic, antiproliferative effect in cultured glomerular mesangial cells. In vascular smooth muscle, ANF inhibits cell proliferation (hyperplasia) as well as cell growth (hypertrophy). ANF has growth-regulatory properties in a variety of other tissues such as brain, bone, myocytes, red blood cell precursors, and endothelial cells. The cellular mechanisms involved in the growth-regulatory actions of ANF are not totally clear. Evidence supports involvement of both biological and clearance receptors for ANF, guanosine 3’,5’-cyclic monophosphate (cGMP) and cGMP-independent mechanisms, and protooncogene expression. For the most part, the actions reviewed in this report represent in vitro phenomena, and it is unclear whether these effects are relevant to normal physiology or pathophysiology. Nevertheless, an emerging hypothesis is developing which states that circulating and autocrine/paracrine factors such as ANF interact to regulate the vasomotor tone and cellular growth of a variety of tissues such as vascular smooth muscle and the glomerulus. natriuretic mesangium; receptor

peptides ; growth substances; adrenal cortex; glomerular vascular smooth m U scle; guanosine 3’,5’-cyclic monophosphate;

WORK of DeBold et al. (18) led to the discovery that supernatants of atria1 homogenates, when injected into bioassay rats, caused a potent natriuresis and diuresis (18). DeBold postulated that atria1 tissue contained a factor, now termed atria1 natriuretic factor (ANF), which had potent natriuretic properties. A great deal of information regarding the structure and function of ANF has been accumulated in the decade since the classic paper of DeBold et al. (for a recent comprehensive review, see Ref. 12). In pharmacological concentrations, the peptide clearly has natriuretic and vasorelaxant properties. It is of interest that the exact mechanisms involved in the natriuretic activity, and also the role of the peptide in the normal physiology of volume homeostasis and blood pressure control, remain controversial (I 1, 28, 29). For the most part, studies dealing with ANF have focused on the role of the peptide in the physiology and pathophysiology of volume homeostasis and blood pressure regulation. However, several lines of reasoning have provided an incentive for investigating a potential role for ANF in cellular growth regulation. 1) ANF binding sites, or ANF-induced guanosine 3’,5’-cyclic monophosphate (cGMP) accumulation, have been shown in numerous tissues that may not be intimately involved in volume homeostasis. For example, ANF binding sites or ANF-induced cGMP accumulation occur in 3T3 fibroblasts, testis, intestine, lung, and liver (7,66). One expla-

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nation for this widespread distribution of ANF binding sites is that some represent biologically silent, clearance receptors (42) rather than biologically active receptors. On the other hand, the peptide may be a circulating hormone or possibly an autocrine/paracrine factor with diverse effects on diverse tissues. 2) Various mitogens have been shown to elevate cytosolic free calcium (48) or stimulate phosphatidylinositol turnover into the watersoluble inositol phosphates in target cells (47). In this regard, ANF has been shown to lower cytosolic calcium (6,32) and inhibit phosphatidylinositol turnover (10) in mesangial cells. Both of these effects would be predicted to be antimitogenic. 3) ANF has been shown to have a number of actions that appear to be antagonistic to the actions of angiotensin II. Recent studies suggest that angiotensin II may induce vascular smooth muscle cell growth (hyperplasia and/or hypertrophy) (27, 49). It was therefore tempting to speculate that ANF might inhibit vascular smooth muscle cell growth. 4) Sporn and Roberts (61) have recently discussed the concept that a variety of peptide growth factors are multifunctional. For example, a given peptide may have both stimulatory and inhibitory effects on cell proliferation, as well as effects unrelated to the control of cell growth. The natriuretic, vasorelaxant, and aldosterone -inhibitory effects of ANF support the concept that ANF is a multifunctional peptide and raise the qu .estion as to a role for the peptide in the co ntrol of cell growth.

0 1992 the American

Physiological

Society

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F911

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EDITORIAL

An increasing body of information relating growth factors to inflammatory and proliferative disease continues to accumulate. For example, the ability of platelet-derived growth factor to stimulate migration and proliferation of smooth muscle cells in culture has suggested that this growth factor may be important in the process of atherosclerosis (3, 55). In addition, loss of responsiveness to or failure to produce growth inhibitors such as transforming growth factor-p may contribute to cancer development (45). Therefore the concept that ANF might possess growth-regulatory properties is potentially of considerable interest. The purpose of this work is to critically review the information that supports a role for ANF in growth regulation and to add some perspective to the subject. A summary of the literature dealing with ANF and cell growth is compiled in Table 1. ADRENAL

GLAND

Early studies demonstrated that the zona glomerulosa of the adrenal gland was a target site for ANF. The basal amount of aldosterone released from rat zona glomerulosa cells, as well as angiotensin II-stimulated aldosterone production, was inhibited by ANF (9). The earliest study dealing specifically with a role for ANF in mitogenesis was performed in adrenal glomerulosa cells. In this study, rat ANF stimulated [ 3H] thymidine incorporation into the DNA of bovine adrenal glomerulosa cells in primary culture (33). The authors commented that the mitogenic effect was a dose-dependent one. However, in fact there was no concentration of ANF at which the effect was not seen. Thymidine uptake was increased by a factor of 2 compared with control at the lowest concentration of ANF tested (lo- l2 M) and by a factor of 4.5 compared with control at the highest concentration of ANF tested ( 10e6 M). These findings, suggesting that ANF possesses promitogenic properties, are at odds with other studies in the adrenal gland (see below). The cause for this discrepancy is not clear, although it should be pointed out that this work utilized bovine tissue, whereas rat tissue has -provided the bulk of information. Two additional pertinent studies deal with the issue of ANF and growth in the adrenal gland (46, 53). These Table 1. Growth-regulatory

properties

REVIEW

studies are somewhat unique in this area in that they were performed as in vivo rather than in vitro investigations. ANF was infused into rats for 7 days via osmotic pumps. The dosage infused was 20 pg. kg-l h-l which should produce pharmacological rather than physiological plasma concentrations. After the 7-day infusion period, these investigators performed morphometric assessments of zona glomerulosa and zona fasciculata (Table 2). As shown, ANF infusion reproducibly caused a notable atrophy of zona glomerulosa cells, mainly due to a decrease in the mitochondrial and smooth endoplasmic reticulum compartments. ANF had no effect on the same morphometric parameters in the zona fasciculata of these rats, suggesting that this was not simply a toxic effect of ANF infusion. Captopril-treated rats showed a reduction in these parameters in zona glomerulosa, which was totally reversed by addition of angiotensin II. Addition of ANF to the captopril/angiotensin II-treated rats again caused growth inhibition. The authors concluded that ANF exerts an inhibitory effect on the growth of rat zona glomerulosa. The mechanism underlying this effect may be direct and does not appear to involve blockade of angiotensin II (a potent stimulator of zona glomerulosa growth). The inhibitory effects of ANF and captopril were not additive. Therefore ANF may interfere with postreceptor events that are similar to those involved in angiotensin II-induced stimulation of rat zona glomerulosa growth. l

GLOMERULUS

Mesangial cells are clearly a target site for ANF. For example, as noted earlier, ANF reduces cytosolic calcium in these cells (6, 32). In addition, ANF inhibits contraction of these cells (8). Furthermore, this cell has properties in common with both vascular smooth muscle cells as well as fixed macrophages (62). Therefore studies dealing with proliferation of these cells may have relevance to proliferative renal diseases, as well as to processes involving proliferation of vascular smooth muscle cells such as atherosclerosis and hypertension. Two groups working independently found similar effects of ANF on rat mesangial cell mitogenesis. Mesangial cells in culture were made quiescent by removal of

of ANF

Tissue

Finding

Adrenal zona glomerulosa (bovine) Adrenal zona glomerulosa (rat) Glomerular mesangial cells (rat) Glomerulus (rat)

Mitogenic effect in cultured cells Growth inhibition with in vivo infusion Antimitogenic effect in cultured cells Stimulation of glomerular growth with in vivo infusion in remnant rat model Antimitogenic effect in cultured cells Antihypertrophic effect in cultured cells Suppression of growth hormone secretion by cultured cells Antimitogenic effect in cultured cells Inhibition of resumption of meiosis in cultured complexes Reduction of cell volume in isolated cells Antimitogenic effect in cultured cells Antimitogenic effect in cultured cells Stimulation of colony formation in culture in presence of EPO Increase in EPO secretion by cultured cells

VSMC (rat, rabbit) VSMC (rat) Anterior lobe pituitary cells (rat) Tibia epiphyseal growth plate cells (avian) Oocyte-cumulus complexes (rat) Atria1 and ventricular myocytes (rabbit) Aortic endothelial cells (bovine) Brain astroglia (rat) Erythroid progenitor cells (human) Renal ANF,

carcinoma atria1

cells (human)

natriuretic

factor;

VSMC,

vascular

smooth

muscle

cells; EPO,

erythropoietin.

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Reference

33 46, 53 4, 5, 37 17 1, 14, 34, 38, 68 34, 35 58 52 63 15 36 40 50 64

EDITORIAL

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Table 2. Effect of ANF on morphometric parameters of rat adrenal zona glomerulosa Group

Volume

of Cells,

pm3

Volume

of Mitochondrial

Compartment,

pm3/cell

Surface

of SER

Membranes,

pm2/cell

Control 644t95 140t22 3,626&522 ANF 452t68* 88&12* 2,344+357” %Decrease 30 37 35 Values are means t SD in 8 separate experiments. Average volume of cells was determined from light micrographs by measuring number of nuclei of parenchymal cells per mm3 of zona. Volume and surface densities of various organelles per cell were determined by utilizing conventional * P < 0.01 vs. control. [Modified from Mazzocchi et al. (46).] stereological procedures. SER, smooth endoplasmic reticulum.

serum-containing medium. They were reactivated to enter the cell cycle by exposure to either 5% serum (37) or a serum-free defined mesangial medium containing insulin, bovine serum albumin, and soybean lipids (4). As a measure of mitogenesis, DNA synthesis was assessed by determination of [ 3H] thymidine incorporation into trichloroacetic acid (TCA) -precipitable material. Addition of ANF was shown to inhibit [3H]thymidine incorporation into the DNA of mesangial cells by ~50%. In these experiments, the half-maximal response to ANF was seen at a concentration as low as lo-l1 M (4). Therefore physiological concentrations of ANF appear to be capable of inhibiting mesangial cell mitogenesis. In mesangial cells exposed to the defined medium plus fibroblast growth factor, [3H] thymidine incorporation was 30% higher than in cells exposed to the defined medium alone. Addition of ANF again inhibited [3H]thymidine incorporation by 50% (4). These experiments indicated a lack of specificity to the inhibitory effect of ANF on mesangial cell growth and suggested an undefined common mechanism of action. ANF was shown to decrease the cross-sectional surface area of rat mesangial cell colonies by 43% (37). Furthermore, in mesangial cells undergoing logarithmic proliferation, ANF reduced cell number by 33% (5). These studies therefore verify that ANF not only inhibits mesangial cell mitogenesis as measured by [3H] thymidine uptake, but also inhibits proliferation of these cells as measured by either cell counts or surface area of cell colonies. Recent work has begun to clarify the cellular mechanisms involved in the antimitogenic effect of ANF in mesangial cells. Figure 1 demonstrates that the inhibitory 100 9 2 90 -E 0 80 0 8 70 g 5‘E

60

g

40

i __-----

3T3 ---____

y-------~

1

50 MC

effect of ANF on rat mesangial cell mitogenesis was a dose-dependent one. This inhibitory effect was not seen in identical experiments performed in 3T3 fibroblast monolayers (5). Unlike mesangial cells, which have biologically active ANF receptors, 3T3 fibroblasts have only biologically silent ANF receptors, termed clearance receptors (42, 51). This work supports the concept that the antimitogenic effect of ANF is cell specific and is mediated via the biological rather than the clearance ANF receptor. However, with regard to the receptor involved, studies described in a later section support a role for the clearance receptor in rat aortic smooth muscle and astroglia (Table 3). There is a rather large body of literature dealing with the effects of cyclic nucleotides on cell proliferation in a variety of tissues. Cyclic nucleotides do appear to play a role in cell proliferation, but the issue is confusing, depending on the tissue, concentration of agent, and the presence or absence of serum and other mitogens. For example, in 3T3 fibroblasts, CAMP is a growth promoter (56). In T lymphocytes both CAMP and cGMP suppressed proliferation (43). Because of the proposed second messenger role of cGMP in a variety of the actions of ANF, studies were performed to explore the role of cGMP in the antimitogenic action of ANF seen in rat mesangial cells. In these studies, [3H] thymidine incorporation was measured in cells exposed to a variety of nitric oxidegenerating vasodilators such as sodium nitroprusside (a nonhormonal agent that stimulates guanylate cyclase independently of receptor-mediated mechanisms) and also exogenous 8-bromo-cGMP (5, 24). These agents caused a dose-dependent inhibition of thymidine incorporation. These studies indicate that cGMP can mimic the antimitogenic effect of ANF in mesangial cells but do not imply causation. For example, the threshold for ANF-induced cGMP accumulation in mesangial cells is at least one order of magnitude greater than the threshold for ANF-induced inhibition of mitogenesis (5). Furthermore, the concentrations of exogenous cGMP required to see an antimitogenic effect were often very high (lo-’ M). At this concentration, there is concern

T 3o wt~ 2

Table 3. Growth-regulatory

I, ,o-12

,o-ll

,o-10

ANF

,o-9

,o-8

,o-7

properties of ANF:

mechanisms involved

(M)

Fig. 1. Dose-response effect of atria1 natriuretic factor (ANF) on [“Hlthymidine incorporation into DNA of mesangial cells (MC) and 3T3 fibroblasts (3T3). Values are percentage of control, where control represents thymidine incorporation in cells reactivated by a defined serum-free medium. Data points are means (error lines are GE) of triplicate determinations in 4-8 different cell strains [reprinted from Apwl (5)l.

Receptor Biological

5, 34 Numbers mechanism.

Subtype Clearance

cGMP Dependent

Independent

14,40 5, 24, 25, 34, 38, 63 5, 14, 26, 60 are specific references with data supportive of indicated See text for details.

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that the exogenous cGMP may cross-react with CAMPdependent protein kinase or may raise intracellular levels of CAMP by inhibiting phosphodiesterase activity (8). In addition, recent preliminary work has demonstrated that, in human mesangial cells, n itric oxide-generating agents caused no changes in intracellular cGMP levels despite effective inhibition of serum and platelet-derived growth factor-induced mesangial cell proliferation (59,60). Interestingly, 3T3 fibroblast cells exposed to pharmacological concentrations of ANF were found to make cGMP (5, 21), but in this cell line there was no inhibition of mitogenesis induced by ANF (5). Finally, a number of nitric oxide-generating vasodilators were found to inhibit mitogenesis in 3T3 fibroblasts despite the fact that they had no effect on cGMP accumulation in these cells, which lack soluble guanylate cyclase activity (26). In summary, while cGMP may play a role in the growth-regulatory properties of ANF, important cGMP-independent mechanisms appear to be involved (Table 3). As noted earlier, various mitogens have been shown to elevate cytosolic free calcium in target cells (48). ANF lowers cytosolic calcium in rat mesangial cells (6,32), and therefore this effect could contribute to the antimitogenic effect. Serum-stimulated mitogenesis is accompanied by elevations in cytosolic free calcium (37). In this setting, ANF decreased resting calcium. Therefore the peak calcium response seen with serum was significantly attenuated (37). Further studies may be necessary to establish a causal link between ANF-induced alterations in intracellular calcium and mitogenesia. Although the bulk of studies support the concept that ANF has antimitogenic properties in mesangial cells, this finding has not been universal. Arginine vasopressin was found to be a mitogen in cultured rat mesangial cells (23). In these studies, neither ANF nor verapamil had any effect on vasopressin-induced mitogenesis as measured by C3H]thymidine incorporation at 24, 48, or 72 h (23). Interestingly, in these same studies, ANF did reduce basal and vasopressin-stimulated cytosolic free calcium. Thus there appears to be a dissociation in these experiments between calcium effects and mitogenesis effects. The studies described above showing an inhibitory effect of ANF on growth of glomerular mesangial cells have utilized in vitro tissue culture techniques. In preliminary work, utilizing in vivo techniques, ANF was conB

Quiescent

ANG

II

Quiescent

ANG

II

Fig. 2. Effect of ANF on [“Hluridine (A) and [3H]leucine (B) incorporation into quiescent and angiotensin II (ANG II)-stimulated rat aortic smooth muscle cells. Open bars represent vehicle; closed bars represent ANF (10e7 M). ANG II concentration was lops M. Data points are means (error lines are &SE) in 8-12 experiments. * P < 0.05 vs. vehicle. [Modified from Itoh et al. (34).]

REVIEW

tinuously infused via subcutaneously placed minipumps in rats subjected to 5/6 nephrectomy (17). Glomerular volume was found to be increased by 20% in remnant rats receiving ANF versus remnant rats not receiving ANF. These studies, undertaken in an attempt to explain the finding that high salt intake results in compensatory hypertrophy in the remnant kidney model, suggest that chronic infusion of ANF may augment glomerular size. Further work will be necessary to confirm these findings and to explain the possible discrepancy between the in vitro and in vivo results. However, this discrepancy and others described in this paper are most likely not simply due to differences in experimental design. The actions of many peptide growth factors depend to a large extent on the context of other signal molecules present, as will be discussed at the conclusion of this report. VASCULAR

SMOOTH

MUSCLE

Because vascular smooth muscle has specific ANF receptors (16, 57), a potential role for ANF in cellular growth regulation in this tissue could have important implications in relation to atherosclerosis and hypertension. Recent evidence suggests that this may be the case. Cultured rat vascular smooth muscle cells exposed to platelet-derived growth factor were shown to incorporate more than twofold the amount of [3H]thymidine compared with cells grown in 2% fetal calf serum alone (1). ANF reduced thymidine incorporation in this setting in a dose-dependent manner. The concentrations of ANF required to inhibit mitogenesis appeared to be in the pharmacological range. For example, 10m7 and 10e6 M ANF inhibited mitogenesis by 25 and 59%, respectively. No attempt was made to investigate the possible mechanisms involved. Recent work has confirmed the antimitogenic effect of ANF in vascular smooth muscle and, for the first time, has shown that ANF also inhibits hypertrophy of vascular smooth muscle cells (34). Although both hyperplasia and hypertrophy of smooth muscle have been emphasized as important in the pathophysiology of hypertension and atherosclerosis, hypertrophy may predominate in the chronic state (20, 34). This work is therefore of considerable interest. These investigators first studied the effect of ANF on [3H]thymidine incorporation into cultured rat aortic smooth muscle cells. ANF inhibited both basal and 1% fetal calf serum-stimulated [ 3H] thymidine incorporation by 55-60%, confirming the antimitogenic action. Rates of protein and RNA syntheses were assessed by determination of [ 3H] leucine and [ 3H]uridine incorporation, respectively, into TCA-precipitable material. As shown in Fig. 2, cells exposed to angiotensin II incorporate more [3H]uridine and [3H]leucine than do quiescent cells, indicating a hypertrophic action of angiotensin II in this setting. ANF inhibited protein and RNA synthesis both in quiescent and angiotensin II-stimulated cells (34). The maximal antihypertrophic action of ANF was -50% and appeared to occur at pharmacological concentrations of ANF. In a similar manner, ANF inhibited RNA and protein synthesis in cells stimulated by transforming growth factor-p, suggesting that the inhibitory effect was a generalized one.

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EDITORIAL

A number of studies were performed to investigate the mechanism of the antihypertrophic action of ANF. A ring-deleted analogue of ANF (C-ANP), which exhibits higher affinity to the clearance receptor rather than the guanylate cyclase-linked biological receptor, was studied. As measured by [3H]uridine incorporation, the antihypertrophic action of the analogue was attenuated in quiescent cells and nonexistent in cells stimulated by angiotensin II (34). This work implies that the inhibitory action appears to be primarily mediated by the guanylate cyclase-linked biological ANF receptor, and not the clearance receptor (Table 3). Interestingly, recent work supports a role for the clearance receptor in mediating the antimitogenic effect of ANF in rat aortic smooth muscle cells (14). In these studies, C-ANP failed to inhibit serum-induced mitogen .esis. However, another linear ANF peptide analogue, also selective for the clearance receptor, did inhibit serum-induced mitogenesis. Furthermore, C-ANP antagonized the antimitogenic effect of the linear analogue as well as the native 28-amino acid ANF. These data suggest that occupation of the clearance receptor by C-ANP blocks the antimitogenic effect, implying that the action may be mediated by the clearance receptor (Table 3). An important issue not addressed by the authors would be the explanation of why C-ANP itself has no antimitogenic effect. The role of cGMP in the antimitogenic and antihypertroph ic action of ANF in rat vascular smooth muscle has been investigated. A variety of nitric oxide-generating vasodilators, which stimulate cGMP accumulation in vascular smooth muscle, have been shown to inhibit mitogenesis as measured by [3H] thymidine incorporation into the DNA of rat aortic smooth muscle cells in culture (25). These nitrovasodilators also inhibited cell proliferation as measured bY counting cell number. Exogenous 8-bromo-cGMP inhibited mitogenesis, as well as RNA synthesis as measured by [ 3H]uridine incorporation (34). In rabbit aortic smooth muscle cells, sodium nitroprusside, &bromo-cGMP, and ANF inhibited serum-induced mitogenesis (38). These experiments suggest that cGMP can mimic the antimitogenic and antihypertrophic actions of ANF in vascular smooth muscle. However, as discussed in detail in the preceding section, they do not prove that cGMP mediates these actions. Indeed, as described above, the linear ANF peptide analogue that inhibited serum-induced mitogenesis did not stimulate cGMP accumulation in rat aortic smooth muscle cells (14). Once again, as in mesangial cells, a role for cGMPindependent mechanisms must be considered (Table 3). Preliminary data have recently been reported which indicate that cultured rat aortic smooth muscle cells synthesize and secrete both mature and NH2-terminally extended forms of ANF (67). This is potentially of great interest in view of the fact that it suggests that ANF may function not only as a circulating hormone but also may have autocrine/paracrine actions. Therefore local concentrations of ANF may reach levels greater than in the plasma, which could lend credence to a number of studies already described in which the actions of ANF were seen only at fairly high concentrations. These same authors have reported additional preliminary data which confirm

F915

REVIEW

that ANF inhibits mitogenesis in vascular smooth muscle and mesangial cells by 55% (68). [Unlike smooth muscle cells, rat mesangial cells have not been shown to synthesize ANF (54). Cells exposed to vehicle, angiotensin II, or arginine vasopressin synthesized no ANF when measured by radioimmunoassay at 6 and 24 h (R. G. Appel, unpublished data) .] Recent advances in the technology of molecular biology and molecular genetics have enabled investigators to begin to probe the molecular events that are important in cellular growth regulation. For example, a number of oncogenes have been associated with various tumor types (19, 22). These genes appear to encode proteins that can function at a variety of levels of growth regulation. In addition, there appear to be antecedent genes, which are called protooncogenes. These genes are normal cellular genes and are involved in the regulation of normal cellular growth or proliferation. Recently, investigators have begun to study the possibility that the growth-regulatory properties of ANF may be mediated by effects of ANF on protooncogene expression. In cultured rat aortic smooth muscle cells, angiotensin II was found to rapidly induce c-fos as well as c-jun and jun-B mRNA expression (35). A number of oncoproteins (fos, jun, and myc) are found in the nucleus (19). Some of these may bind specific sequences of DNA and are thought to regulate the transcription of genes whose products ultimately cause cellular division (19). ANF at high concentrations had no effect on the expression of the protooncogenes induced by angiotensin II as noted above (35). The investigators therefore concluded that the inhibitory effect of ANF on the growth of vascular smooth muscle cells in vitro did not occur through the regulation of these specific protooncogene expressions. This, however, does not rule out the possibility that ANF has effects on other protooncogenes that were not studied in this investigation. In addition, it is of considerable interest that certain growthinhibitory signals such as those encoded by the retinoblastoma gene have also been recently identified (19). These genes have been termed antioncogenes and may function to restrain or confine normal cellular proliferation, because inactivation of these specific genes appears to trigger cancer formation (22). Therefore it would be of interest to study the potential role of ANF in the induction of antioncogene expression. OTHER

TISSUES

In addition to the work described above, there have been a number of studies in a variety of tissues that may have relevance to the potential role of ANF as a modulator of growth. For example, in cultured anterior lobe cells of rat pituitary, ANF has been shown to suppress basal and growth hormone-releasing factor-induced secretion of growth hormone (58). These findings could suggest an indirect role for ANF in cell growth via the regulation of growth hormone secretion. ANF has been shown to inhibit basal and parathyroid hormone-stimulated [ 3H] thymidine incorporation into the DNA of cultured avian tibia epiphyseal growth plate cells (52). In the same study, ANF had no effect on proliferation of avian skin fibroblasts. ANF was found to enhance epidermal growth factor receptor mRNA in

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EDITORIAL

these cells (31). In this setting, epidermal growth factor is a mitogen, suggesting that the two peptides interact to modulate proliferation of these cells. ANF slightly reduced E3H]thymidine incorporation into the DNA of cultured fetal rat calvaria (65). However, this reduction was not found to be statistically significant. ANF caused a dose-dependent inhibition of spontaneous maturation in rat oocyte-cumulus complexes (63). ANF and cGMP appeared to exhibit an inhibitory role in the control of the meiotic process in rat oocytes. Recently, investigators studied the effect of ANF on cell volume of isolated rabbit atria1 and ventricular myocytes (15). In these studies, myocyte dimensions were determined by means of video microscopy techniques. In pharmacological concentrations, ANF was found to decrease the volume of both atria1 and ventricular cells under isotonic and hypotonic conditions. Interestingly, this effect was blocked by bumetanide, suggesting that the mechanism by which ANF reduces cell volume might involve inhibition of the Na+-K+-ZClcotransporter system. Recent preliminary work indicates that ANF at pharmacological concentrations inhibits serum-stimulated mitogenesis of cultured bovine aortic endothelial cells (36). In these studies, ANF attenuated the gene expression of basic fibroblast growth factor, which acts as a local promoter of endothelial cell growth. Furthermore, the inhibitory effect of ANF was eliminated by blocking endogenous production of basic fibroblast growth factor, suggesting a newly proposed mechanism by which ANF may exert an inhibitory effect on growth . These studies, along with preliminary data already described indicating that aortic smooth muscle cells synthesize and secrete ANF (67), demonstrate the potential importance of local communication between the endothelium and underlying smooth muscle. ANF, as well as brain natriuretic peptide, has now been shown to inhibit mitogenesis in rat brain astroglia (40), with no effect seen in bovine brain capillary endothelial cells. These effects were seen at concentrations of native ANF as low as 10BIO M. In addition, similar findings were seen with C-ANP, which binds only the clearance receptor. Therefore these studies again suggest a role for the clearance receptor in the growth-regulatory action s of ANF. A summary of all the studies reviewed in this manuscript leads to some confusion regarding which ANF receptor is linked to the growth- regulatory action (Table 3). Although both the biological and clearance receptors may be involved, recent studies point to the clearance receptor and suggest a new function for this receptor. This is of particular interest because the majority of ANF receptors in the kidney and vasculature are clearance receptors (2, 42). As noted earlier, peptide growth factors are often multifunctional. In fact, the same factor may be mitogenie or antimitogenic depending on the tissue and the presence or absence of other signal molecules. In this regard, although ANF appears to be antimitogenic in most systems, the peptide may stimulate human lymphocyte proliferation (30) and appears to potentiate human erythroid colony formation in culture (50). In the pres-

REVIEW

ence of erythropoietin, ANF caused a dose-dependent stimulation of both early and late erythroid precursor cells (burst-forming unit erythroid cells and colony-forming unit erythroid cells). Also of interest in this regard, human ANF caused a dose-related increase in erythropoietin secretion from cultured human renal carcinoma cells (64). SUMMARY

AND

CONCLUSIONS

Recent evidence supports the notion that ANF has growth-regulatory properties. These properties, along with the well-known natriuretic and vasorelaxant effects, indicate that ANF is a multifunctional peptide, similar to a number of peptide growth factors (61). The concept that a variety of peptide growth factors are multifunctional may reflect the fact that certain early cell signals mediate a variety of different actions. For example, increases in cytosolic calcium may eventuate in cell contraction or cell mitogenesis or cell secretion, depending on the setting. In general, the data summarized in this report support the concept that ANF inhibits cell proliferation (hyperplasia) as well as cell growth (hypertrophy). Cytotoxicity studies indicate that this is not a toxic effect of ANF. The mechanisms involved are controversial (Table 3), but recent data support a role for the clearance receptor and cGMP-independent mechanisms. With only a few notable exceptions, these studies have utilized in vitro techniques, in particular tissue culture. It should be stressed that it is unclear whether these in vitro phenomena are relevant to normal physiology or pathophysiology. Despite the fact that the majority of studies support the concept that ANF has growth-inhibitory properties, there are several discrepancies in the literature that have been described in this review. These discrepancies are most likely due to the context of other signal molecules present. Important factors that determine context include culture conditions, the concentration of the particular agent, and the presence or absence of other growth factors. Receptor subtype switching may occur when tissues are grown in culture. The state of differentiation of the target cell may also be of importance (61). In addition, it appears that receptor occupation by a peptide growth factor may alter the cellular distribution or the binding affinity of a receptor for a second peptide growth factor (61) On the basis of the data presented, an emerging hypothesis is developing, which states that circulating and autocrine/paracrine factors such as ANF interact to regulate the vasomotor tone and cellular growth of a variety of tissues such as vascular smooth muscle and the glomerulus. The actions of endothelial cell products such as endothelium-derived relaxing factor and endothelin in modulation of vasomotor tone and growth regulation support this concept (13, 39, 41, 44). Future work might be designed to attempt to define a potential counteracting role for ANF in modulating hypertensive vascular changes and progressive glomerular disease. I thank Karen Bullard for manuscript preparation. Work from this laboratory was supported by a grant-in-aid American Heart Association, North Carolina Affiliate.

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from

the

EDITORIAL Address School of 27157-1053.

for reprint Medicine,

requests: Medical

Dept. Center

of Medicine, Bowman Blvd., Winston-Salem,

Gray NC

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