Ghrelin improves the fine structure of atrial natriuretic factor (ANF) granules and intercalated disc junctions in experimentally-induced myocardial infarction in rats / La grelina mejora las estructuras finas de los gránulos de factor natriurético atrial (ANF) y las uniones intercaladas de disco en el infarto de miocardio inducido experimentalmente en ratas

Ghrelin is a novel growth hormone-releasing peptide administered to treat myocardial infarction (MI). However, the underlying mechanism of its protective effects against MI remains unclear. A total of sixty healthy Sprague Dawley male rats were included. The first one is the sham-operated control group were the rats that underwent the same surgical used to induce MI but without tying the left anterior descending coronary artery (LAD) and received normal saline (0.5 ml) as vehicle; the second MI model group were rats with LAD ligation and received normal saline (0. 5 ml) and the third one is MI+ghrelin group were rats that were exposed to surgery to induce MI but received ghrelin (100 µ/kg, orally, 2x/day). At the end of the experiment after 21 days post-MI, rats were sacrificed and processed for ultrastructural demonstration. Our experiment showed that ghrelin inhibited cardiomyocyte apoptosis. Concomitant administration of ghrelin with MI treated rats of this study appeared to show a considerable protection of the atrial tissues. This study revealed that the sarcoplasm was occupied by normal myofibrils with clear striations and others appeared with minor disruption. Normal distribution of atrionatriuretic factor (ANF) granules and well preserved mitochondrial integrity (preserved cristae, normal size and shape), nucleus chromatin arrangement and striated pattern of clear bands (Z and H) compared to the MI group. Intact intercalated disc with clear identification of fully formed fascia adherence and desmosomes with a reconstruction of gap junction (nexus) was also noticed. Atrial myocytes after myocardial infarction is often associated with subsequent heart failure, which could lead to a fatal outcome. In a rat model of experimental myocardial infarction, peripheral ghrelin administration attenuated myocyte dysfunction, well-preserved desmosome, adherent and gap junction of the intercalated disc and normally distributed ANF granules.

La grelina es un nuevo péptido liberador de hormona de crecimiento administrado para tratar el infarto de miocardio (IM). Sin embargo, el mecanismo subyacente de sus efectos protectores contra el IM aún no se conocen. Se incluyeron un total de 60 ratas macho Sprague Dawley saludables. En el grupo control se incluyeron ratas que fueron sometidas a una cirugía utilizada para inducir el IM, pero sin ligar la arteria coronaria descendente anterior izquierda (ACDAI) y recibieron suero fisiológico normal (0,5 ml) como vehículo; el segundo grupo modelo de IM fueron ratas con ligadura de ACDAI y recibieron suero fisiológico normal (0,5 ml); el tercer grupo estuvo formado por ratas con IM + grelina, expuestas a la cirugía para inducir IM pero luego recibieron grelina (100 m/kg, oralmente, 2x/día). Al final del experimento, 21 días después del infarto de miocardio, los animales fueron sacrificados y procesados para el estudio ultraestructural. Nuestro experimento mostró que la grelina inhibe la apoptosis de los cardiomiocitos. La administración concomitante de grelina en ratas con IM parece indicar una protección considerable de los tejidos atriales. Además, el estudio reveló que el sarcoplasma estaba ocupado por miofibrillas normales con estriaciones claras y otras con una alteración menor. Se encontró una distribución normal de los gránulos del factor natriurético atrial (FNA) e integridad mitocondrial bien conservada (crestas conservadas, tamaño y forma normales), disposición de la cromatina del núcleo y patrón estriado de bandas claras (Z y H) en comparación con el grupo IM. También se observó un disco intercalado intacto con una clara identificación de la adherencia de la fascia completamente formada y desmosomas con una reconstrucción de la unión gap (nexo). Los miocitos atriales, después de un infarto de miocardio, a menudo se asocian con insuficiencia cardíaca posterior, que podría conducir a un desenlace fatal. En un modelo de rata de infarto de miocardio experimental, la administración de grelina periférica atenuó la disfunción de miocitos, con conservación del desmosoma, adherencia y unión de la brecha del disco intercalado y una distribución normal de los los gránulos de FNA.

[Chromogranin A: immunocytochemical localization in secretory granules of frog atrial cardiomyocytes].

Chromogranin A (CgA) is a member of the granin family of acidic proteins that present in the secretory granules (SGs) of many endocrine, neuroendocrine and neuronal cells. Atrial natriuretic peptide (ANP)-storing SGs in atrial cardiomyocytes of rat heart also contain CgA. Cardiosuppressive effect of CgA-derived peptides (vasostatins) on in vitro isolated and perfused working frog and rat hearts has been shown under both basal conditions and beta-adrenergic stimulation. More recently it has been revealed that rat heart produces and processes CgA-derived vasostatin-containing peptides. Until now nothing has been known about the presence of CgA in an amphibian heart. We have investigated the subcellular localization of CgA in atrial myocytes of adult frog Rana temporaria heart using ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP has shown that out of three morphologically different types (A, B and D) of specific cytoplasmic granules (SCGs) present in myocytes only two (A and B)--large (120-200 nm in diameter) granules with more and with less electron dense core--exhibit immunoreactivity (IR) to these two antigens. The third type (D) of granules (80-100 nm in diameter) are small membrane bound granules characterized by highly electron dense core surrounded with a thin halo. These granules revealed negative reaction on immunostaining for both CgA and ANP. The presence of CgA- and ANP-IR in the same SCGs in frog atrial myocytes is consistent with the endocrine nature of these granules. Taking into account our and literature data we propose that CgA present in frog atrial cardiomyocite SCGs might be a precursor of vasostatin-containing peptides, as it takes place in rat heart. It is possible that these CgA-derived peptides together with ANP exert their regulatory function through the autocrine and/or paracrine mechanisms and play important cardioprotective role in frog heart under stress condition.

Fluorescence imaging microscopy of cellular markers in ischemic vs non-ischemic cardiomyopathy after left ventricular unloading.

BACKGROUND: The heart undergoes repair and initiates protective mechanisms via ventricular unloading. We examined the presence of 2 markers in pre-unloaded and post-unloaded human cardiac tissue that are important indicators of cardiac failure, tumor necrosis factor-alpha and inducible nitric oxide synthase. We also measured 2 nuclear transcription factors, NFkappaB50 and NFkappaB65, comparing quantities and localizations to determine if mechanical unloading reduced their presence, as these markers are also thought to be indicators of impending heart failure. Amounts and localizations in patients that had been diagnosed with either ischemic or non-ischemic cardiomyopathy were compared after mechanical unloading with a left ventricular assist device. To establish that unloading had been achieved, levels of atrial natriuretic protein were determined. METHODS: Core biopsies were harvested at assist device implantation and removal. Fluorescence deconvolution microscopy image reconstructions of fluorescence probes were correlated with data obtained by western Blot and electrobility shift assays. RESULTS: Statistically significant differences in localization and amounts of tumor necrosis factor and nitric oxide synthase were seen between pre- and post-assist device samples. Amounts of tumor necrosis factor and nitric oxide synthase in ischemic tissue were increased at the time of assist device removal, but decreased in dilated or idiomyopathic samples. Ventricular unloading resulted in reduced levels of natriuretic protein, with the greatest reduction being seen in ischemic tissue. Both NFkappaB50 and NFkappaB65 increased in ischemic tissue, but only NFkappaB50 in non-ischemic samples. CONCLUSIONS: Changes in localization of the factors and altered levels of cytokine and nitric oxide synthase indicate that the heart switches to a "protective and repair" mode, and mechanical unloading allows this transition to occur. Observed changes were dependent on the etiology of the disease.

Molecular identification and immunohistochemical localization of atrial natriuretic peptide in the heart of the dromedary camel (Camelus dromedarius).

Atrial and B-type natriuretic peptide (ANP and BNP) are cardiac hormones synthesized and secreted by the myoendocrine cells of the heart. They exert potent actions on body fluid balance. Since various body organs including the heart are under high physiological stress during water and food deprivation in the desert nomads, we intended to perform molecular biological and histological studies of ANP in the heart of the dromedary camel Camelus dromedarius. Initially, we isolated cDNAs encoding ANP from the atrium and BNP from the atrium and ventricle of the dromedary camel. Putative mature ANP, deduced from the cDNA sequence, was identical to that of human and pig ANP, but the putative mature BNP was more diverse and was most similar to pig BNP (94% identity). Thus, we used antisera raised against human ANP that did not cross-react with pig BNP in the subsequent immunohistochemical studies. The ANP-expressing myoendocrine cells are most concentrated in the right atrium, to a lesser extent in the left atrium, and almost absent in the left ventricle. The immuno-positive cells are scattered uniformly in each region and are characterized by the presence of immunoreactive granular deposits around the nucleus. The left atrium comprises some ramifications of conductive cells (Purkinje fibers), some of which also contained ANP-immunoreactive granules. At the electron microscopic level, myoendocrine cells possessed secretory granules primarily in the perinuclear zone and a well-developed Golgi apparatus. The present study is the first comprehensive report dealing with the molecular cloning and immunohistochemical localization of ANP in the heart of a desert dwelling mammal.

The effect of poly(ethylene glycol)-poly(D,L-lactic acid) diblock copolymers on peptide acylation.

The combination of poly(ethylene glycol) (PEG) with a biodegradable poly(ester), such as poly(D,L-lactic acid) (PLA), is an approach that has been successfully used for the stabilization of proteins and peptides in several biodegradable delivery devices. The acylation of peptides inside degrading PLA microspheres has been described only recently as another instability mechanism related to the accumulation of polymer degradation products inside eroding PLA. We investigated whether the block copolymerization of PLA with PEG reduces peptide acylation inside degrading microspheres. Diblock copolymers consisting of poly(D,L-lactic acid) covalently bound to poly(ethylene glycol)-monomethyl ether (Me.PEG-PLA) were used for these investigations. Human atrial natriuretic peptide (ANP) was incorporated into microspheres manufactured from Me.PEG5-PLA45, a diblock copolymer with an overall PEG content of 10%. Peptide integrity inside the microspheres was monitored by HPLC-MS analysis during 4 weeks of microsphere degradation in isotonic phosphate buffer (pH 7.4) at 37 degrees C. Inside the degrading Me.PEG5-PLA45 microspheres, acylation products as well as an oxidation product of ANP were formed. The results demonstrate that the combination of PEG with PLA does not necessarily display a favorable effect concerning peptide acylation inside degrading polymer microspheres. However, they also suggested that the acylation reaction is mainly driven by the formation and accumulation of polymer degradation products inside the degrading microspheres.

Cardiac rhabdomyomas exhibit a fetal pattern of atrial natriuretic peptide immunoreactivity.

Rhabdomyomas are the most common heart tumors seen in infancy. However, whether they represent hamartomas or true neoplasms derived from cardiomyocytes is still controversial. The fetal pattern of atrial natriuretic peptide (ANP) expression (predominant in the atrial and ventricular subendocardium) becomes altered during the early postnatal period to that typical of the adult (all atrial cardiomyocytes and some cells in the ventricular impulse-conducting system). To better comprehend the nature and origin of cardiac rhabdomyomas, we investigated the immunohistochemical expression of ANP in seven surgically excised ventricular specimens and two necropsy cases of multiple, atrial, and ventricular rhabdomyomas in children aged 1 to 34 days. Immunogold labeling for ANP at the ultrastructural level was also performed on three ventricular tumors. Although all atrial tumors were immunoreactive for ANP, these usually showed a variable number of faintly positive cardiomyocytes, contrasting with the diffuse and intense immunoreactivity of the surrounding atrial myocardium. ANP was detected in the ventricular tumors of five (56%) of the nine cases. The positive ventricular tumor cells predominated in the subendocardium and areas with prominent fibrous tissue, usually around blood vessels. Immunoelectron microscopy of the ventricular tumors demonstrated rare, positive cytoplasmic granules surrounded by membranes, usually located near the nuclei. We conclude that cardiac rhabdomyomas exhibit a fetal pattern of ANP immunoreactivity, which suggests delayed maturation of the tumoral cardiomyocytes, reinforcing the notion that cardiac rhabdomyomas are fetal hamartomas.

[The study of ultrastructure of atrial natriuretic peptides immunoreactive in cochlear of guinea pigs].

To further explore ultrastructure of atrial natriuretic peptides immunoreactive (ANP-IR) products in cochlear of guinea pig, and secretory type, ultrastructure of ANP-IR products of stria vascularis were studied with immunoelectronic microscopy. The result indicated: ANP-IR granules were seen in the cytoplasm, more obviously at the ends of the nucleus. There were two types of granule: storage and secretory granule. The study suggests that ANPs were secreted by rupture of restriction membrane, not by exocytosis secretion.

Ultrastructural study of atrial specific granules in experimental renovascular hypertension elicited by the constriction of both renal arteries.

The heart has an endocrine activity which depends on the secretion of a natriuretic, diuretic and hypotensive factor contained in osmophilic, secretory granules localized in the myocardiocytes and called "atrial specific granules" (the atrial natriuretic factor, ANF). In this paper, the relationship between these specific granules and renovascular hypertension elicited by the constriction of both renal arteries was investigated at the electron microscope level during the acute, subacute and chronic phases of hypertension. Male Wistar CHbb THOM rats were divided in three groups: 1) clipped rats; 2) sham operated rats; 3) ether anesthesia as unique manoeuver 48 h before decapitation. Blood pressure increased progressively after the constriction of both renal arteries. The atrial specific granules were not affected by ether anesthesia alone; 48-72 h after clipping the granules almost disappeared and this situation persisted up to the 6th week. In sham operated rats the picture was very similar to the clip rats 48 and 72 h after surgery (severe granule disappearance); in contrast, at one, two and six weeks after surgery, the granularity of cardiomyocytes in sham rats was absolutely restored. It is concluded that: 1) similarities in morphology of atrial specific granules in sham and clip rats 48 and 72 h after surgery would suggest that stress plays a primary role in determining the observed images; 2) thereafter, the contrast between sham and clip rats 1, 2 and 6 weeks after surgery would indicate that the ANF is linked to the subacute and chronic regulation of renovascular hypertension.

Ultrastructural study of atrial specific granules in experimental renovascular hypertension elicited by the constriction of both renal arteries

El corazón tiene una actividad endocrina que depende de la secreción de un factor nafriurético, diurético e hipotensor contenido en gránulos osmiófilos secretorios localizados en los miocardiocitos y llamados " granulos atriales específicos" (atrial natriuretic factor, ANF). En este trabajo se investigó al microscópio electrónico la relación existente entre los gránulos atriales específicos y la hipertensión renovascular provocada por constricción de ambas arterias renales, examinándose los períodos agudo, subagudo y crónico de hipertensión. Se usaron ratas macho, Wistar CHbb Thom. Los animales fueron divididos en tres grupos: 1) ratas con compresión bilateral de la arteria renal; 2) ratas con operación simulada; 3) ratas con anestesia con éter como única maniobra, 48 hs antes de la decapitación. La presión arterial aumentó progresivamente después de la constricción de las arterias renales. Los gránulos atriales específicos no fueron afectados por la anestesia con éter. Por el contrário 48-72h después de la compresión de las arterias renales los gránulos atriales específicos prácticamente desaperecieron y esta situación persistía 1, 2 y 6 semanas después. En las ratas con operación simulada se observó un cuadro similar a las ratas con constricción arterial (severa desaparición de los gránulos) pero 1, 2 y 6 semanas más tarde la granularidad de los cardiomiocitos se había restaurado completamente. Se concluye: 1) la similitud de la respuesta en ratas con constricción de ambas arterias renales y en ratas con operación simulada 48-72 h después de la intervención sugiere que el estrés desempeña un papel inicial en los resultados observados. 2) el contraste observado 1, 2 y 6 semanas después entre ratas con constricción arterial pemanente y ratas con operación simulada indicaría que el factor natriurético atrial está vinculado con la regulación subaguda y crónica de la hipertensión renovascular

Ultrastructural study of atrial specific granules in experimental renovascular hypertension elicited by the constriction of both renal arteries

El corazón tiene una actividad endocrina que depende de la secreción de un factor nafriurético, diurético e hipotensor contenido en gránulos osmiófilos secretorios localizados en los miocardiocitos y llamados " granulos atriales específicos" (atrial natriuretic factor, ANF). En este trabajo se investigó al microscópio electrónico la relación existente entre los gránulos atriales específicos y la hipertensión renovascular provocada por constricción de ambas arterias renales, examinándose los períodos agudo, subagudo y crónico de hipertensión. Se usaron ratas macho, Wistar CHbb Thom. Los animales fueron divididos en tres grupos: 1) ratas con compresión bilateral de la arteria renal; 2) ratas con operación simulada; 3) ratas con anestesia con éter como única maniobra, 48 hs antes de la decapitación. La presión arterial aumentó progresivamente después de la constricción de las arterias renales. Los gránulos atriales específicos no fueron afectados por la anestesia con éter. Por el contrário 48-72h después de la compresión de las arterias renales los gránulos atriales específicos prácticamente desaperecieron y esta situación persistía 1, 2 y 6 semanas después. En las ratas con operación simulada se observó un cuadro similar a las ratas con constricción arterial (severa desaparición de los gránulos) pero 1, 2 y 6 semanas más tarde la granularidad de los cardiomiocitos se había restaurado completamente. Se concluye: 1) la similitud de la respuesta en ratas con constricción de ambas arterias renales y en ratas con operación simulada 48-72 h después de la intervención sugiere que el estrés desempeña un papel inicial en los resultados observados. 2) el contraste observado 1, 2 y 6 semanas después entre ratas con constricción arterial pemanente y ratas con operación simulada indicaría que el factor natriurético atrial está vinculado con la regulación subaguda y crónica de la hipertensión renovascular (AU)

Fine structure of atrial natriuretic peptide (ANP)-granules in the atrial cardiocytes in the hamster, guinea pig, rabbit, cat and dog.

In the hamster, guinea pig, rabbit, dog and cat, the right and left atria and ventricles were examined by immunohistochemistry, and the right auricular cardiocytes were studied by transmission electron microscopy. Moreover, ANP-granules in the cardiocytes were analyzed by ultrastructural morphometry. Immunohistochemically, the most intensely ANP-reactive cardiocytes were localized in the right auricle, particularly more prominent in the hamster and guinea pig than in the rabbit, dog and cat. The immunoreaction in the dog and cat was weaker than that in the rabbit. ANP-immunoreactivity was not detected in the ventricular myocardium of any of all species examined, but was occasionally observed in the subendocardium of the ventricular septum. Ultrastructurally, ANP-granules were localized principally in the perinuclear region associated with the Golgi apparatus and scattered throughout the sarcoplasmic layers. The Golgi apparatus of the cardiocytes was better developed in the hamster and guinea pig than in the rabbit, dog and cat. It was poorly-developed in the dog and cat. By ultrastructural morphometry, the number of granules was greatest in the hamster followed by the guinea pig, rabbit and dog or cat, in this order. On the other hand, the diameter of granules was largest in the guinea pig and reduced via the hamster to the rabbit. The diameter was significantly smaller in the dog than in the rabbit. The diameter of granules of the cat was lay between the rabbit and dog.

Localization of brain and atrial natriuretic peptide in human and porcine heart.

We have compared the localization of brain and atrial natriuretic peptide-like immunoreactivity in human and porcine hearts, using immunohistochemical techniques at both the light and ultrastructural level and specific antisera to amino-(cardiodilatin) and carboxy-terminal regions of the atrial natriuretic precursor molecule and to brain natriuretic peptide. Atrial myocardial cells in human fetal, normal adult and failing explanted hearts, displayed immunoreactivity for both brain and atrial natriuretic peptide-like sequences. At the subcellular level, brain natriuretic peptide-, cardiodilatin- and alpha-atrial natriuretic peptide-like immunoreactivity were co-localized to secretory granules in atrial myocardial cells. Immunoreactivity was also detected in the left (64%) and right ventricular free walls (23%) of 22 failing explanted hearts, but not in donor cardiac tissues. A gradient of natriuretic peptide immunostaining was observed across ventricular free walls and immunoreactivity for both natriuretic peptide sequences co-localized to secretory granules in a subpopulation of myocardial cells, concentrated in subendocardial regions of the ventricular walls. Brain and atrial natriuretic peptide-like immunoreactivity were also demonstrated in porcine atrial myocardium and cells of the ventricular conduction system. The parallel distribution of cardiac brain and atrial natriuretic peptide-like immunoreactivity suggests a dual regulation and co-storage of the natriuretic peptides in human and porcine hearts.

Atrial natriuretic peptide (ANP)-granules of auricular cardiocytes in aging Mongolian gerbils.

The atrial natriuretic peptide (ANP)-granules of right auricular cardiocytes were studied in aging Mongolian gerbils by immunohistochemistry and transmission electron microscopy, and analyzed by ultrastructural morphometry. Immunohistochemically, the ANP immunoreactions were weaker in 3 and 4-year-old animals than in 90-day-old, 1- and 2-year-old animals. There was no difference in the reaction among 90-day-old, 1 and 2-year-old animals, or between 3 and 4-year-old animals. Ultrastructurally, the morphological features in 1 and 2-year-old animals were similar to those of 90-day-old animals, but distinct in 3 and 4-year old animals by the presence of a few lysosomal structures in the cell. By ultrastructural morphometry, number and diameter of ANP-granules of 3 and 4-year-old animals were significantly smaller than those in the 90-day-old, 1 and 2-year-old. There was no significant difference in number and diameter among the 90-day-old, 1 and 2-year-old, or between the 3 and 4-year-old.

Conformation in solution of porcine brain natriuretic peptide determined by combined use of nuclear magnetic resonance and distance geometry.

The conformation in solution of porcine brain natriuretic peptide was determined by combined use of NMR spectroscopy and distance geometry. A set of 157 inter-proton-distance constraints was derived from the two-dimensional NOE spectra, and further a set of three hydrogen bond constraints was obtained from analysis of the temperature dependence of labile protons. The five structures with minimal violations were selected after performing distance-geometry calculations starting from 40 random initial conformations. The distance-geometry structures were further refined by the use of restrained energy minimization and restrained molecular dynamics. This structure shows a compact conformation with the carboxy-terminal region, Asn21-Tyr26, folded back to the disulfide-linked loop region, Cys4-Cys20. The characteristics of the conformation determined are as follows: conformations of the three segments interposed by glycine residues, which are Arg7-Ile12, Ser14-Leu18 and Cys20-Arg25, were well defined and the segments Arg7-Ile12 and Cys20-Arg25 are rather close to each other and nearly parallel. The biological significance of these local conformations is discussed on the basis of comparisons with those of atrial natriuretic peptide reported by Kobayashi et al.

Localization and characterization of the N-terminal fragment of atrial natriuretic factor (ANF) precursor in the frog heart.

The localization of the N-terminal fragment of the atrial natriuretic factor (ANF) precursor in the heart of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques using an antiserum directed against synthetic rat ANF (Asp11-Ala37). At the optic level, positive material was found in most atrial myocytes. Staining of consecutive sections of frog heart with antibodies against N-terminal and C-terminal regions of the proANF molecule showed that both peptides are contained in the same cardiocytes. In the rat atrium, antibodies against the N-terminal ANF region induced a more intense labeling than in the frog atrium. Electron microscopic studies indicated that all secretory granules present in frog atrial cardiocytes contain N-terminal ANF-like immunoreactive material. The positive material localized in frog atrium was characterized by gel filtration and radioimmunological detection. Serial dilutions of frog atrial extracts exhibited displacement curves which were parallel to that obtained with synthetic human ANF (Asn1-Asp30). Sephadex G-50 gel chromatography of the immunoreactive material showed that the N-terminal ANF-like immunoreactivity eluted in a single peak corresponding to high molecular weight material. These results indicate that the N-terminal fragment of frog proANF is immunologically and biochemically related to the homologous mammalian peptide.

Urodilatin and beta-ANF: binding properties and activation of particulate guanylate cyclase.

Urodilatin (ANF-(95-126] and beta-ANF, the antiparallel dimer of ANF-(99-126), are naturally occurring members of the ANF family. We studied their receptor binding properties in human platelets and Triton-solubilized membranes from bovine adrenal cortex and their ability to activate particulate guanylate cyclase in bovine adrenal cortex. In human platelets containing R2-receptors not coupled to particulate guanylate cyclase urodilatin binds with similar affinity as ANF-(99-126) (KD: 55 pM), whereas beta-ANF has an affinity lower than the truncated ANF-(103-123) (KD: 295 pM and 154 pM). Scatchard analysis indicates one binding site for urodilatin as well as for beta-ANF. In adrenal cortex containing predominantly R1-receptors coupled to particulate guanylate cyclase, urodilatin binds with a higher affinity (KD: 30 pM) than ANF-(99-126) (KD: 52 pM) and stimulates to a similar extent to ANF-(99-126) (about two fold at 1 muM), whereas beta-ANF has a smaller affinity (KD: 120 pM) and stimulates particulate guanylate cyclase to a lower extent than ANF-(99-126). The data from platelets and adrenal cortex show that beta-ANF has low binding affinities but stimulates particulate guanylate cyclase, whereas urodilatin appears to be a physiological R1-agonist.