Cellular and Molecular Neurobiology, Vol. 9, No. 1, 1989

Commentary

Discovery of Atrial Natriuretic Factor in the Brain: Its Characterization and Cardiovascular Implication

Tadashi Inagami, 1 Issei Tanaka, ~ J. C. McKenzie, 1 Mitsuaki Nakamaru, 1 Ryoichi Takayanagi, ~ Teruaki Imada, ~ Roland Pochet , 2 A . Resibois, 2 Mitsuhide Naruse, 3 Kiyoko Naruse, 3 and Tamotsu Shibasaki 3

Received July 15, 1988; accepted July 16, 1988

KEY WORDS: atrial natriuretic factor (ANF) in brain; radioimmunoassay; immunohistochemistry; ANF mRNA; ANF molecular forms; antidipsogenic action; antihypertensive action; suppression of vasopressin.

SUMMARY

1. We have devised a radioimmunoassay for atrial natriueretic factor (ANF). Its application to rat brain extract led to the discovery of ANF in the brain. In addition to the hypothalamus and the pontine medullary region, it was widely distributed.

2. ANF in the brain is stored in a low molecular weight form, in contrast to pro-ANF in the atria. Thus, the processing of pro-ANF in the bran neuronal cells is different from that in the atria.

3. ANF was found in the anterior and posterior lobes of the pituitary, the peripheral ganglia, adrenergic neurons, and the adrenal medulla.

4. Brain ANF suppressed stimulated dipsogenesis, basal and stimulated vasopressin release, and angiotensin II-stimulated pressor effects.

5. ANF in the peripheral neuronal system inhibits catecholamine synthesis and release. Thus, central ANF functions to reduce the peripheral fluid volume and vascular tone in concert with the peripheral ANF.

X Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.

2 Department of Histology, Free University of Brussels, Brussels, Belgium. 3 Department of Internal Medicine, Tokyo Women's College, Shinjuku-ku, Tokyo, Japan.

75

0272-4340/89/0300-0075506.00/0 © 1989 Plenum Publishing Corporation

76 Inagami et al.

INTRODUCTION

The discovery by de Bold et al. of a potent natriuretic activity in the rat atrial extract prompted investigators to look for its mechanism of action and led them to the discovery of vasorelaxing activity of atrial extract (Currie et al. , 1983; Koike et al., 1984). Whether the natriuretic and vasorelaxant activities are elicited by the same peptide was investigated by us. We found that the two activities copurify and concluded that the atrial natriuretic factor (ANF) has vasorelaxant activity (Grammer et al. , 1984).

DETERMINATION OF ANF IN THE BRAIN AND PLASMA

Structure determination (Misono et al. , 1984) and subsequent synthesis (Sugiyama et al. , 1984) of the low molecular weight ANF permitted us to develop a radioimmunoassay of ANF in plasma and tissues (Tanaka et al. , 1984). Using the immunoassay methods, we and Gutkowska et al. (1984) demonstrated that ANF is a circulating hormone.

Like various gut peptides, we found immunoreactive ANF in the brain. The hypothalamus and the pontine-medullary region were found to contain the highest concentration of ANF (Tanaka et al. , 1984). lmmunohistochemical studies indicated the presence of immunoreactive ANF in the paraventricular

Fig. 1. Immunoreactive ANF in the paraventricular nucleus and the outer layer of the median eminence of the rat.

ANF in Brain 77

nucleus and the outer layer of the median eminence (Fig. 1) and in axons with varicosities in the thalamus (Fig. 2). These findings ruled out the possible contamination of plasma ANF in the radioimmunoassay of ANF in the brain.

The concentration of ANF in the brain is very low. The highest concentra- tion (10-20/tg/g wet tissue) found in rat hypothalamus is 10,000-100,000 times less than that in rat atria (Tanaka et al. , 1984; Morii et al. , 1985; Zamir et al. , 1986; Imada et al. , 1985). Morii et al. (1985) surveyed various rat brain regions by microdissection and radioimmunoassay and found that the hypothalamus and septum are the highest in ANF concentration, followed by the midbrain, cortex, olfactory bulb, thalamus, and pontine medullary region. Zamir et al. used punches from frozen sections to determine the regional distribution of ANF in the rat brain. ANF was found to be present extensively in a variety of rat brain regions, in circumventricular organs, telencephalic, diencephalic, and mesen- cephalic regions, medulla oblongata, and spinal cord. These regions include the thalamus, hypothalamus, hippocampus, amygdala, dorsal raphe nucleus, locus coeruleus, periaquaductal gray, organum vasculosum of the lamina terminalis, subfornical organ, and area postrema (Zamir et al. , 1986).

Immunohistochemical studies support these observations. The ANF- containing neurons were localized in preoptic and hypothalamic neurons (Jacobo- witz et al., 1985; Saper et al. , 1985). Detailed studies combining immunohis- tochemical localization and radioimmunoassay with microdisected brain tissues have been reported by Kawata et al. (1985). Neurons that were prominently stained by antibodies to ANF were the ventral region of the lateral septal nucleus, periventricular preoptic nucleus, red nucleus of the stria terminalis,

Fig. 2. Immunoreactive ANF in the neuronal axon in the thalamus of the rat.

78 Inagami et al.

periventricular and dorsal regions of the paraventricular nucleus, ventromedial nucleus, dorsomedial nucleus, arcuate nucleus, median mamillary nucleus, and periaquaductal gray matter. Scattered immunopositive nuclei were in the cingu- late cortex, endopiriform nucleus, lateral hypothalamic area, and pretectal and dorsal thalamic areas. Immunoreactive varicose fibers were seen in the glomeru- lar layer of the olfactory bulb, in the external layer of the median eminence, central to the paramedian regions of the interpeduncular nucleus, and in the paraventricular hypothalamic nucleus. Considerable numbers of immunoreactive varicose fibers were also observed in the globus pallidus, medial and central amygdaloid nuclei, dorsal raphe nucleus, dorsal parabrachial nucleus, locus coreruleus, vagal dorsal motor nucleus, solitary nucleus, subformical organ, and organum vasculosum laminae terminalis.

The neuronal localization of ANF in extensive regions of the brain indicated local synthesis of ANF in the brain. This concept was strengthened by the demonstration of ANF mRNA in the brain extract by us (Inagami et al., 1987) (Fig. 3) and in various regions such as the hypothalamus, brain stem, cerebellum, cerebral cortex, and thalamus by Gardner et al. (1987).

In addition to the neuronal tissues of the brain, McKenzie et al. (1985) discovered the presence of ANF immunoreactivity in the ovoid cells of the anterior pituitary [most likely luteinizing hormone (LH) gonadotrophs] and adrenal medulla. These findings were supported by Inagaki et al. (1986a); Inagami et al., (1987) and Gardner et al. (1986). ANF was reported to inhibit the release of proopiomelanocortin-derived peptides from anterior and intermediate lobes of the pituitary (Shibasaki et al., 1986b). One of the functions of the anterior pituitary ANF may be such a paracrine function.

1353 1078 872_ 603

Fig. 3. Northern blot analysis of rat brain ANF mRNA (left lane) compared with rat atrial ANF mRNA (right lane).

ANF in Brain 79

Gutkowska et al. (1987) found immunoreactive ANF in the rat posterior pituitary. In addition, ANF was found in the peripheral nervous system such as autonomic ganglia (Inagaki et al. , 1986b; Morii et al. , 1987; Dubinski et al., 1987a), autonomic nervous system (Debinski et al. , 1986), and cerebrospinal fluid (Marumo et al., 1987). Colocalization of ANF in acetylcholine and substance P-containing neurons has been reported (Standaert et al. , 1986).

MOLECULAR PROPERTIES OF BRAIN ANF

The molecular properties of ANF in the neuronal tissues were examined. We have quickly treated rat brain in a boiling acidic solution, homogenized the tissue, and examined ANF in the extract by reverse-phase and size-separation high- performance liquid chromatography (HPLC). The immunoreactive ANF in the brain extract was exclusively of the lower molecular weight type (tr-ANP), in contrast to the atrial ANF, which was entirely of the high molecular weight form (Imada et al. , 1985). Glembotsky et al. (1985) reported similar observations. Shiono et al. (1986) characterized the rat brain ANF by reverse-phase HPLC and found that it elutes at positions where ANF(102-126) and ANF(lo3_a26) (25 and 24 amino acid residue peptides, respectively) emerge. Ueda et al. (1987) isolated ANF from porcine brain and determined the amino acid sequence of these peptides, which were identified as ANF(102_126) and ANF(1o3-126). These observa- tions indicate that brain ANF is shorter than the circulating form of ANF, which contains 28 residues (Thibault et al. , 1985; Schwartz et al., 1985). As shown in Fig. 3, the enzyme responsible for the processing of proANF in the brain must be different from that in the atrium.

Recently a new peptide BNP (brain natriuretic peptide) was isolated from porcine brain (Sudoh et al. , 1988). This peptide shows actions similar to those of ANF (Schwartz et al. , 1988) and has a structure highly homologous to that of ANF (Sudoh et al. , 1988) (Fig. 4). However, it does not cross-react with antibodies to rat or human ANF. While it was isolated from the brain, it seems to be present in atrium in a sizable quantity and share the same receptors in peripheral tissues (unpublished observation of our laboratory).

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Fig. 4. The amino acid sequences of ANF and BNP. Arrow A indicates the cleavage site for the generation of plasma ANF99 126, with 28 amino acid residues. Arrows B indicate the cleavage sites for the activation of pro-ANF in the brain neurons. ANF, atrial natriuretic factor; BNP, the natriuretic peptide isolated from porcine brain.

80 Inagami et al.

SECRETION OF BRAIN ANF

In an in vitro experiment with a depolarizing concentration of KCI, we showed that ANF is released in organ culture medium in the presence of calcium ion (Tanaka and Inagami, 1986; Shibasaki et al., 1986a). Marumo et al. (1987) reported the observation that sodium ion may be one of the regulators of hypothalamic release of ANF. These findings, together with the presence of ANF mRNA and intraneuronal localization of ANF immunoreactivity, indicated that ANF is synthesized in the brain, then released, and exerts its effect locally on neuronal receptors, and some of the effect is transmitted to the periphery by humoral and neuronal mechanisms. In addition to the local action of endogenous ANF, the bloodborne ANF can also exert its effect through circumventricular receptors.

These observations indicated that in the brain, ANF is stored in the processed form and released upon specific release signal. This is in contrast to the atrium, in which pro-ANF, with 126 amino acid residues, is stored in atrial storage granltles and the activation process takes place at the time of the secretion of ANF.

FUNCTIONS OF BRAIN ANF

The wide distribution of ANF in various regions of the brain suggests the versatile action of ANF. In view of the observations that the various physiological responses elicited by ANF in peripheral systems often antagonize the actions of angiotensin II, many investigators explored the possible reactions of ANF on

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ANF in Brain 81

centrally mediated hypertensive, dipsogenic, and hypophysical hormone releasing actions of angiotensin II.

Intracerebroventricularly administered ANF was found to inhibit water drinking in nonstimulated rats and rats stimulated by angiotensin II or water deprivation (Antunes-Rodriguez et al., 1985; Nakamura et al., 1985; Masotto and Negro-Vilar, 1985; Nakamaru et al., 1986) (Fig. 5). Peripheral administration of ANF was also found to inhibit the dipsogenesis (Antunes-Rodriguez et al., 1985). Since ANF did not affect the food intake, this antidipsogenic action is considered as a specific action of ANF (Masotto and Negro-Vilar, 1985). It is likely that these effects are mediated by ANF receptors in circumventricular organs (Quirion et al., 1988).

Obana et al. (1985) have shown in vitro effects of ANF on vasopressin secretion from superfused rat anterior pituitary. The basal level of secretion as well as angiotensin II stimulated or potassium-stimulated vasopressin secretion was almost completely inhibited by 10-1°M ANF.

Dehydration and hemorrhage induce secretion of vasopressin. Samson (1985) has shown that intracerebroventricular ANF inhibits vasopressin release in rats induced by these stimuli. In v ivo (Takahashi et al., 1981) and in vitro studies supported the inhibitory effects of ANF on vasopressin secretion. Centrally administered ANF was also found to inhibit salt preference in rat (Antunes- Rodriguez et al., 1986; Itoh et al., 1988). The inhibition of vasopressin secretion observed in these studies negates earlier reports of the stimulation of vasopressin release by ANF (Januszewicz et al., 1985). Taken together, these observations strongly indicate that ANF interacts with central receptors and elicits responses all directed toward the reduction of the extracellular fluid volume.

Intracerebroventricular angiotensin II induces hypertensive responses in the peripheral circulation. ANF icv was found to attenuate such a response (Itoh et aI., 1986). While the ANF effect is rapidly lost if the pretreatment with ANF is performed 20-30 min prior to angiotensin administration (Nakamaru et al.,

Fig. 6. Antihypertensive effects of icv ANF aga- inst icv angiotensin II (Ang II) in pentobarbitol- anesthetized Sprague-Dawley rats. ANF (5/~g) was administered 4min prior to icv ANgl I (100 ~g).

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82 Inagami et al.

1986a), at shorter intervals ANF completely suppressed the angiotensin-induced hypertension under anesthesia (Fig. 6).

Another interesting central effect of ANF is the inhibition of vasopressin neurons reported by Standaert et al. (1987), which is in line with the inhibitory effects of ANF on other actions of vasopressin. ANF was also reported to inhibit the central dopaminergic neurons (Nakao et al. , 1986).

ANF also has effects on peripheral nervous systems. We found that ANF suppresses the release of norepinephrine evoked by electrical stimulation from mesenteric nerve endings (Nakamaru and Inagami, 1986b). Debinski et al. (1987b) found that it partially inhibits the synthesis of catecholamine in superior cervical ganglia. This, ANF attenuates vascular tone by its direct action on the vascular tissues as well as by its inhibitory action on the peripheral neuronal system. Adrenomedullary chromaffin cells contain a considerable amount of ANF (McKenzie et al. , 1985). While its function is not clear, its secretion is stimulated by carbachol (unpublished results of our laboratory).

CENTRAL ANF IN HYPERTENSION

Although the exact role of brain ANF in the pathophysiology of various diseases is not understood, Takayanagi et al. (1986a) and Morii et al. (1988) found that ANF levels in the hypothalamus, pons, and septum are markedly elevated in spontaneously hypertensive rats. ANF levels are comparable in young prehyper- tensive rats, but the ANF content in these tissues increases as the hypertension progresses, just like plasma ANF levels of spontaneously hypertensive rats (SHR) (Imada et al. , 1988). One of the intracellular mediators of ANF action, cyclic GMP, is generated by particulate guanylate cyclase-containing ANF receptors.

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ANF in Brain 83

Interestingly, ANF-sensitive particulate guanylate cyclase activity in the brain stem (Fig. 7) and hypothalamus of SHR is markedly elevated (Zamir etal., 1986; Takayanagi et al., 1986b). The physiological meaning of the increased guanylate cyclase activity is not clear.

ACKNOWLEDGMENTS

The authors thank Jeanne Williams for valuable assistance in the preparation of the manuscript. This work was supported by USPHS Research Grants HL 14192 and HL 35323.

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