Autoradiography of atrial natriuretic peptide (ANP) receptors in the rat brain.

Quantitative autoradiography was used to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat brain and to study their regulation. Peptide receptors are selectively located to circumventricular organs outside the blood brain barrier, such as the subfornical organ, and to brain areas involved in fluid and cardiovascular regulation. Dehydration, either by water deprivation of normal rats, or chronic dehydration present in homozygous Brattleboro rats lacking vasopressin, results in large increases in ANP binding in receptor number in the subfornical organ. In the deoxycorticosterone acetate (DOCA)-salt hypertensive model, only salt treatment, but not DOCA alone or the combination of DOCA-salt, increased the ANP receptor number in the subfornical organ and the choroid plexus. Both young and adult genetically hypertensive rats have a greatly decreased ANP receptor number in the subfornical organ and the choroid plexus. Selective displacement with an inactive analog lacking the disulfide bond (ANP 111-126) suggests that genetically hypertensive rats may lack C (clearance) atrial natriuretic peptide receptors. Our results implicate brain atrial natriuretic peptide receptors in the central response to alterations in fluid regulation and blood pressure.     

Alterations in atrial natriuretic peptide receptors in rat brain nuclei during hypertension and dehydration

We have studied the localization, kinetics, and regulation of receptors for the circulating form of the atrial natriuretic peptide (99-126) in the rat brain. Atrial natriuretic peptide receptors were discretely localized in the rat brain, with the highest concentrations in circumventricular organs, the choroid plexus, and selected hypothalamic nuclei involved in the production of the antidiuretic hormone vasopressin and in blood pressure control. Spontaneously (genetic) hypertensive rats showed much lower numbers of atrial natriuretic peptide receptors than normotensive controls in the subfornical organ, the area postrema, the nucleus of the solitary tract, and in the choroid plexus. These changes are in contrast with those observed for receptors of angiotensin II, another circulating peptide with actions opposite to those of the atrial natriuretic peptide. In acute dehydration after water deprivation, as well as in chronic dehydration such as that present in homozygous Brattleboro rats, there was an up-regulation of atrial natriuretic peptide receptors in the subfornical organ. Thus, circumventricular organs contain atrial natriuretic peptide receptors that could respond to variations in the concentration of circulating peptide. The localization of atrial natriuretic peptide receptors and the alterations in their regulation present in hypertensive and dehydrated rats indicate that these brain receptors are related to fluid regulation, including the secretion of vasopressin, and to cardiovascular function. Atrial natriuretic peptide receptors in the choroid plexus may be related to the formation of cerebrospinal fluid.     

Different effects of chronic Na+, Cl−, and K+ depletion on brain vasopressin mRNA and plasma vasopressin in young rats

1. We studied the effects of selective chronic dietary sodium, chloride, or potassium depletion in young rats on vasopressin mRNA levels in the supraoptic and paraventricular nuclei, an index of vasopressin formation, and in plasma vasopressin levels, an index of vasopressin release. 2. All diets significantly increased plasma renin activity, contracted the extracellular fluid volume, and decreased serum osmolarity. 3. In the supraoptic nucleus, vasopressin mRNA levels were significantly decreased in the low-sodium group but were not significantly affected by chloride depletion. 4. There were no significant changes in vasopressin mRNA in the paraventricular nucleus after sodium or chloride dietary depletion. 5. After 2 weeks of potassium depletion, vasopressin mRNA levels were decreased in the supraoptic nucleus. When potassium depletion was prolonged for 3 weeks, vasopressin mRNA levels increased in both supraoptic and paraventricular nuclei. 6. Plasma vasopressin levels were high in animals subjected to dietary chloride depletion or to 3 weeks of potassium depletion. Dietary sodium depletion or 2 weeks of dietary potassium depletion did not significantly affect plasma vasopressin. 7. Our results show that chronic sodium, chloride, or potassium depletion differentially affect brain vasopressin mRNA and vasopressin release in young rats. 8. The effect of these diets may be mediated through changes in the extracellular fluid volume, serum osmolarity, and/or renin angiotensin system.     

Increased atrial natriuretic peptide (6-33) binding sites in the subfornical organ of water deprived and Brattleboro rats

Binding sites for rat atrial natriuretic peptide (6-33) (ANP) were quantitated in the subfornical organ of chronically dehydrated homozygous Brattleboro rats unable to synthesize vasopressin; heterozygous Brattleboro rats, their controls, Long Evans rats and Long Evans rats after 4 days of water deprivation. Brain sections were incubated in the presence of 125I-ANP and the results analyzed by autoradiography coupled to computerized microdensitometry and comparison to 125I-standards. Brattleboro rats and water deprived Long Evans rats presented a higher number of ANP binding sites than their normally hydrated controls. Our results suggest a role of ANP binding sites in the subfornical organ in the central regulation of fluid balance and vasopressin secretion.     

Regulation of angiotensin II binding sites in the subfornical organ and other rat brain nuclei after water deprivation

1. Binding sites for angiotensin II have been localized in forebrain and brain-stem areas of water-deprived and control Sprague-Dawley rats, employing autoradiography with computerized microdensitometry. 2. Angiotensin II receptor sites were identified in the organum vasculosum of the lamina terminalis, subfornical organ, paraventricular nucleus, median preoptic nucleus, area postrema, nucleus of the solitary tract, and inferior olive. 3. After dehydration a significant increases in the concentration of angiotensin II receptors was detected only in the subfornical organ. Although there was an increased concentration of angiotensin II binding sites in the organum vasculosum of the lamina terminalis, the median preoptic nucleus, and the paraventricular nucleus after dehydration, these changes did not reach statistical significance. Other brain nuclei investigated did not show differences in angiotensin II binding sites in the dehydrated rats compared to controls. 4. These results indicate that angiotensin II receptors in the subfornical organ may play an important role in fluid homeostasis during dehydration.     

Renal effects of dopamine and ANP in high volume expanded rats

Both dopamine (DA) and atrial natriuretic peptide (ANP) have been postulated to exert similar effects on the kidney, participating in the regulation of body fluid and sodium homeostasis. In the present study, experiments were performed in anesthetized and isotonic sodium chloride volume expanded rats. After acute volume expansion at 15 % of body weight during 30 min, glomerular filtration rate, urine output, sodium excretion, fractional sodium excretion, proximal and distal sodium excretion and blood pressure were measured. In additional groups we administered ANP or haloperidol or the combination of both to volume expanded animals. Blockade of DA receptors with haloperidol, attenuated diuretic and natriuretic responses to volume load. Proximal sodium excretion was not modified by haloperidol in all experimental groups of rats. Reduction in distal tubular excretion was induced by haloperidol in saline infusion expanded rat but not in ANP treated expanded animals. In conclusion, when exaggerated volume expansion is provoked, both DA and ANP exert renal tubular events, but ANP have a major central role in the regulation of renal sodium handling.     

The role of estradiol in adrenal insufficiency and its interaction with corticosterone on hydromineral balance

Estradiol (E2) plays an important role in controlling the homeostasis of body fluids. Several studies have reported the involvement of the hypothalamic pituitary adrenal axis (HPA) in the homeostatic control of hydromineral balance and the influence of estrogens on the modulation of this system. Nevertheless, until now, the physiological relevance of HPA axis activity on the hydromineral balance in females has not yet been fully elucidated. Therefore, the objective of the present study was to evaluate the effects of E2 (20 μg/animal) pretreatment on neuroendocrine and hydroelectrolyte changes induced by adrenalectomy (ADX) with or without glucocorticoid hormone replacement (corticosterone, CORT; 10 mg/kg) in ovariectomized rats (OVX). The results show that sodium appetite, natriuresis and the elevated plasma angiotensin II (ANG II) concentration induced by ADX were attenuated by E2 pretreatment. Additionally, a reduction of AT1 mRNA expression in the subfornical organ (SFO) and an increase in plasma atrial natriuretic peptide (ANP) concentrations by E2 pretreatment were observed. E2 pretreatment reversed the reduction in water intake induced by ADX in ADX CORT-replaced rats. Moreover, E2 pretreatment attenuated corticotropin releasing factor (CRF) mRNA expression in the paraventricular nucleus (PVN) induced by ADX. In contrast, E2 pretreatment increased CRF mRNA expression in the PVN in ADX CORT-replaced rats. Taken together, these results suggest that E2 has an important role in the modulation of behavioral and neuroendocrine responses involved in the maintenance of body fluid homeostasis in ADX rats with or without glucocorticoid replacement therapy.     

Overlapping distributions of receptors for atrial natriuretic peptide and angiotensin II visualized by in vitro autoradiography: morphological basis of physiological antagonism

Atrial natriuretic peptides exert actions on many key organs involved in blood pressure and water and electrolyte balance. Many of these actions result in a physiological antagonism of angiotensin. To investigate the morphological basis of this interaction, we have mapped the distribution of receptors for atrial natriuretic peptide and angiotensin II in a number of target organs, using 125I-labelled rat atrial natriuretic peptide (99-126) and 125I-labelled [Sar1,Ile8]angiotensin II. In the kidney both atrial natriuretic peptide and angiotensin II receptors were observed overlying glomeruli, vasa recta bundles (high densities), and the outer cortex (moderate density). In the other tissues studied, atrial natriuretic peptide and angiotensin II receptors were codistributed in the adrenal zona glomerulosa, cerebral circumventricular organs including the subfornical organ, organum vasculosum of the lamina terminalis and area postrema, and the external plexiform layer of the olfactory bulb. The concurrent distribution of specific receptors for both peptides at these sites provides the basis for atrial natriuretic peptide to exert a functional antagonism of the actions of angiotensin II on blood pressure and water and electrolyte homeostasis at multiple sites.     

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).     

Atriopeptin prevents angiotensin II-stimulated glucose utilization in the subfornical organ

Previous studies have shown that angiotensin II (ANG II) increases glucose utilization in the subfornical organ and stimulates drinking behavior. We investigated with the deoxyglucose method whether atriopeptin III, an atrial natriuretic peptide (ANP), would prevent this enhanced glucose metabolism and interfere with the drinking response in the presence of ANG II. Two rat models with high circulating levels of ANG II were studied: the homozygous Brattleboro and ANG II-infused Sprague-Dawley rats. ANP decreased the normally enhanced glucose utilization in the subfornical organ in the Brattleboro rat and inhibited ANG II-stimulated glucose metabolism in the subfornical organ of Sprague-Dawley rats. This effect was accompanied by decreased ANG II-stimulated water intake. These findings indicate that ANP may act at the level of subfornical organ to antagonize the dipsogenic action of ANG II.     

Estradiol potentiates hypothalamic vasopressin and oxytocin neuron activation and hormonal secretion induced by hypovolemic shock

Estrogen receptors are located in important brain areas that integrate cardiovascular and hydroelectrolytic responses, including the subfornical organ (SFO) and supraoptic (SON) and paraventricular (PVN) nuclei. The aim of this study was to evaluate the influence of estradiol on cardiovascular and neuroendocrine changes induced by hemorrhagic shock in ovariectomized rats. Female Wistar rats (220-280 g) were ovariectomized and treated for 7 days with vehicle or estradiol cypionate (EC, 10 or 40 μg/kg, sc). On the 8th day, animals were subjected to hemorrhage (1.5 ml/100 g for 1 min). Hemorrhage induced acute hypotension and bradycardia in the ovariectomized-oil group, but EC treatment inhibited these responses. We observed increases in plasma angiotensin II concentrations and decreases in plasma atrial natriuretic peptide levels after hemorrhage; EC treatment produced no effects on these responses. There were also increases in plasma vasopressin (AVP), oxytocin (OT), and prolactin levels after the induction of hemorrhage in all groups, and these responses were potentiated by EC administration. SFO neurons and parvocellular and magnocellular AVP and OT neurons in the PVN and SON were activated by hemorrhagic shock. EC treatment enhanced the activation of SFO neurons and AVP and OT magnocellular neurons in the PVN and SON and AVP neurons in the medial parvocellular region of the PVN. These results suggest that estradiol modulates the cardiovascular responses induced by hemorrhage, and this effect is likely mediated by an enhancement of AVP and OT neuron activity in the SON and PVN.     

Ventricular, paraventricular and circumventricular structures involved in peptide-induced satiety

Cholecystokinin, bombesin or gastrin (2 microliter of 50 ng/microliter) was injected stereotaxically into the paraventricular nucleus of the hypothalamus, the arcuate/ventromedial area, the subfornical organ, the area postrema and the cerebral aqueduct of Sprague-Dawley rats and the effects of these injections on food and water intake were studied. While the injection of cholecystokinin reduced food intake when it was injected into both hypothalamic loci, food and water intake were most severely affected by the injection of this peptide into the cerebral aqueduct. Bombesin reduced food intake after its injection into all areas except the subfornical organ and reliable reductions in water intake were seen after injection of this peptide into all areas except the paraventricular nucleus. Minor reductions in food intake were seen following gastrin injection into the paraventricular nucleus while increased water consumption was observed after this peptide was injected into the paraventricular nucleus and cerebral aqueduct. In a second study 6-hydroxydopamine injections (2 microliter of 8 micrograms/microliter were made into the five areas studied 10 days before animals were injected with 100 micrograms/kg of cholecystokinin (i.p.). All 6-hydroxydopamine-injected animals reduced their food and water intake in response to the cholecystokinin challenge as did intact controls. These results indicate that while the changes in food and water intake produced by the central injection of cholecystokinin, bombesin or gastrin may involve central catecholamine systems, those occurring after its systemic administration do not. Therefore, if the release of gastrointestinal peptides during natural feeding is part of a homeostatic mechanism regulating hunger and satiety, this mechanism may operate without directly involving central catecholamine systems.     

Attenuation of negatively regulated ANP secretion by calcium in hypertrophied atria

Abnormal intracellular Ca(2+)-handling has been described in various heart diseases associated with cardiac hypertrophy. The crucial role of Ca(2+) in the excitation-secretion coupling in atrial cardiomyocytes is not well established. To investigate modulation of atrial natriuretic peptide (ANP) secretion regulated by Ca(2+) in hypertrophied atria, responsiveness of stretch-induced ANP to Ca(2+) was studied using isolated perfused quiescent hypertrophied rat atria. Male Sprague-Dawley rats were given a single subcutaneous injection of 50 mg/kg monocrotaline (MCT) and were sacrificed at 5-6 weeks. In isolated perfused hypertrophied right atria from MCT rats, changes in atrial volume induced by increased atrial pressure caused proportional increases in mechanically stimulated extracellular fluid (ECF) translocation and stretch-induced ANP secretion. Stretch-induced ANP secretion was markedly increased by the depletion of extracellular Ca(2+). However, an accentuation of stretch-induced ANP secretion by Ca(2+) depletion was markedly attenuated in hypertrophied right atria, as compared to control right atria. Therefore, stretch-induced ANP secretion in terms of ECF translocation by Ca(2+) depletion in hypertrophied atria was significantly lower than in control right atria. However, no significant differences were observed between nonhypertrophied and control left atria. Depletion of extracellular Ca(2+) caused a decrease in intracellular calcium in single beating atrial myocytes, which was significantly attenuated in hypertrophied atrial myocytes. The results suggest that attenuation of Ca(2+)-induced negative regulation of ANP secretion in hypertrophied atria may be due to the disturbance of intracellular Ca(2+) regulation.     

Atrial natriuretic peptide (ANP) system in frog skin

In this study the ultrastructure of Rana esculenta skin is described. Cytochemical methods were used to localize guanylate cyclase in the presence of atrial natriuretic peptide and immunocytochemical methods showed the presence of the atrial natriuretic peptide in various levels of skin. The peptide is mainly found in the epithelium and in the lymph sacs of the tela subcutanea. Its receptors are located in the same zones and are indicated by guanylate cyclase activity. We demonstrate that frog skin is a target organ for atrial natriuretic peptide and propose that, at this level, the peptide carries out an important osmoregulatory role.     

Inhibition of dehydration-induced water intake by glucocorticoids is associated with activation of hypothalamic natriuretic peptide receptor-A in rat

Atrial natriuretic peptide (ANP) provides a potent defense mechanism against volume overload in mammals. Its primary receptor, natriuretic peptide receptor-A (NPR-A), is localized mostly in the kidney, but also is found in hypothalamic areas involved in body fluid volume regulation. Acute glucocorticoid administration produces potent diuresis and natriuresis, possibly by acting in the renal natriuretic peptide system. However, chronic glucocorticoid administration attenuates renal water and sodium excretion. The precise mechanism underlying this paradoxical phenomenon is unclear. We assume that chronic glucocorticoid administration may activate natriuretic peptide system in hypothalamus, and cause volume depletion by inhibiting dehydration-induced water intake. Volume depletion, in turn, compromises renal water excretion. To test this postulation, we determined the effect of dexamethasone on dehydration-induced water intake and assessed the expression of NPR-A in the hypothalamus. The rats were deprived of water for 24 hours to have dehydrated status. Prior to free access to water, the water-deprived rats were pretreated with dexamethasone or vehicle. Urinary volume and water intake were monitored. We found that dexamethasone pretreatment not only produced potent diuresis, but dramatically inhibited the dehydration-induced water intake. Western blotting analysis showed the expression of NPR-A in the hypothalamus was dramatically upregulated by dexamethasone. Consequently, cyclic guanosine monophosphate (the second messenger for the ANP) content in the hypothalamus was remarkably increased. The inhibitory effect of dexamethasone on water intake presented in a time- and dose-dependent manner, which emerged at least after 18-hour dexamethasone pretreatment. This effect was glucocorticoid receptor (GR) mediated and was abolished by GR antagonist RU486. These results indicated a possible physiologic role for glucocorticoids in the hypothalamic control of water intake and revealed that the glucocorticoids can act centrally, as well as peripherally, to assist in the normalization of extracellular fluid volume.     

Increased dithiothreitol-insensitive,type 2 angiotensin II receptors in selected brain areas of young rats

1. Angiotensin II receptors have been studied by quantitative autoradiography in selected brain areas of young (2-week-old) and adult (8-week-old) rats. 2. In young rats, angiotensin II receptors were present in brain areas which did not express receptors in the adult brain, such as thalamic nuclei, cortical areas, and the cerebellum. 3. Young rats had more angiotensin II receptors in the subfornical organ than adult rats. In the inferior olive, the number of angiotensin receptors in young animals was 10 times higher than that in adult rats. Angiotensin II binding in the inferior olive was insensitive to incubation in the presence of dithiothreitol. 4. Conversely, the number of angiotensin II receptors in the nucleus of the solitary tract was lower in young rats compared to adults. Incubation in the presence of dithiothreitol resulted in a more than 90% inhibition of angiotensin II binding in the nucleus of the solitary tract. 5. Our results indicate the presence of two types of angiotensin II receptor in brain, one sensitive (type 1) and one insensitive (type 2) to the reducing agent dithiothreitol. 6. The expression of type 2 angiotensin II receptors, insensitive to dithiothreitol, is more marked in young rats, indicating a role for this type of angiotensin receptors in brain development.     

Bilateral acute heart atrial auriectomy reduced diuresis and natriuresis following hypertonic sodium load in anaesthetized dogs

Anaesthetized dogs were deprived of a portion of the atrial natriuretic factor producing tissue by bilateral acute heart atrial auriectomy. Their ability to respond by diuresis and natriuresis either to the expansion of extracellular fluid volume with isotonic saline (3% b.w.) or to hyperosmolality induced by hypertonic saline loading (0.13% b.w. of 20% NaCl solution) was subsequently reduced by about 50%. It is thus suggested that atrial natriuretic system may also play a role in osmoregulation by taking part in the promotion of renal sodium excretion.     

Effects of changes in water-sodium balance on levels of atrial natriuretic factor messenger RNA and peptide in rats

Responses of atrial mRNA, atrial peptide and plasma peptide of atrial natriuretic factor (ANF) to treatments to alter fluid volume were studied in rats using RNA dot hybridization assay and radioimmunoassay. Specific changes in the level of ANF mRNA relative to total atrial RNA were observed in atria from sodium restricted rats and water deprived then sodium loaded rats, demonstrating an association of change in water-sodium balance with the expression of ANF gene. The levels of mRNA and the immunoreactive ANF in plasma decreased to 30% and 15% of controls, respectively, on water-deprivation and then increased again to control levels after administering 1.8% NaCl solution, whereas atrial immunoreactive ANF increased to about twice the control on water-deprivation and decreased again after supplying NaCl solution, in parallel with the level of the hematocrit. These findings suggest that atrial ANF content is dependent more on ANF release than on biosynthesis.     

Differential effects of intravenous hyperosmotic solutes on drinking latency and c-Fos expression in the circumventricular organs and hypothalamus of the rat

Hyperosmotic intravenous infusions of NaCl are more potent for inducing drinking and vasopressin (AVP) secretion than equally osmotic solutions of glucose or urea. The fact that all three solutes increased cerebrospinal fluid osmolality and sodium concentration led the investigators to conclude that critical sodium receptors or osmoreceptors for stimulating drinking and AVP secretion were outside the blood-brain barrier (BBB) in the circumventricular organs (CVOs). We tested an obvious prediction of this hypothesis: that all three solutes should increase c-Fos-like immunoreactivity (Fos-ir) inside the BBB, but that only NaCl should increase Fos-ir in the CVOs. We gave intravenous infusions of 3.0 Osm/l NaCl, glucose, or urea to rats for 11 or 22 min at 0.14 ml/min and perfused the rats for assay of Fos-ir at 90 min. Controls received isotonic NaCl at the same volume. Drinking latency was measured, but water was then removed. Drinking consistently occurred with short latency during hyperosmotic NaCl infusions only. Fos-ir in the forebrain CVOs, the subfornical organ, and organum vasculosum laminae terminalis was consistently elevated only by hyperosmotic NaCl. However, all three hyperosmotic solutes potently stimulated Fos-ir in the supraoptic and paraventricular nuclei of the hypothalamus inside the BBB. Hyperosmotic NaCl greatly elevated Fos-ir in the area postrema, but even glucose and urea caused moderate elevations that may be related to volume expansion rather than osmolality. The data provide strong support for the conclusion that the osmoreceptors controlling drinking are located in the CVOs.     

Identification of an endogenous protease that processes atrial natriuretic peptide at its amino terminus

We have partially purified a thiol-dependent protease from bovine atrial tissue that cleaves the Arg98-Ser99 bond of rat natriuretic peptide (Gly96-Tyr126) to produce the natriuretic Ser99-Tyr126 peptide (cardionatrin I). This was the only hydrolytic product we detected. The existence of the atrial natriuretic peptide system implicates the mammalian heart as an endocrine organ which participates in the hormonal regulation of extracellular fluid volume, electrolyte balance and vascular tone. This enzyme appears to be part of that system. The atrial protease also hydrolyzes the Arg-2-Napthylamide bond of natriuretic peptide stand-in substrates; on the basis of relative Vmax/Km as a measure of substrate specificity, Bz-Leu-Arg-Arg-2-Napthylamide (NA) greater than Bz-Leu-Arg-2-NA greater than Arg-2-NA. There is little or no cleavage between the Arg-Arg pair of the first substrate. Since in the Gly96-Tyr126 peptide the Arg-Arg pair is not the principle cleavage site for this enzyme, it is very unlikely that it is a principle cleavage site for this enzyme in pro-atrial natriuretic factor. It is possible that it is a cleavage site for a different enzyme or the pair may serve as a signal for cleavage at Arg98.