Decoding the molecular switches of natriuretic peptides which differentiate its vascular and renal functions.

Heart failure (HF) is associated with high morbidity and mortality. Dysfunction of blood pressure and/or volume homeostatic processes result in lower perfusion and/or congestion. Treatment strategies exerting differential effects on pressure and volume mechanisms are critical in handling patients with HF. Atrial natriuretic peptides (ANPs) are a key hormone in maintaining circulation. It binds to NP receptor-A (NPR-A) on vasculature, kidneys and nervous system to lowers blood pressure and volume. It exerts a concentration-dependent pharmacological activity, and only increased renal excretion of water and sodium at low doses and vasodilation along with renal effects at slightly higher doses. Recently, we showed that K-Ring (conserved ring of krait venom NP) elicited only vasodilatory properties despite its ability to evoke NPR-A. Through systematic analysis of the structure-function relationships of K-Ring, we have delineated the molecular switches that control vasodilatory and diuretic properties of NPs in anesthetized rats. In the process, we have identified residues that - (a) differentiate vascular and renal functions, (b) affect heart rate and pulse pressure, (c) exhibit sustained effect on vasodilatory function and (d) forceful diuresis switches. Furthermore, we have shown these residues to have equivalent effects on ANP scaffold, thereby introducing modularity in designing function-based ANP analogs. By comparing the ability of designed NPs to evoke cGMP levels, we propose a hypothetical mechanism for the observed tissue-specific effects. The present study opens new avenues in the development of suitable therapeutic agents for personalized care for HF patients.

Recombinant production, purification and characterization of vessel dilator in E. coli.

Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.

Validez y utilidad del péptido ventricular natriurético tipo B (BNP) en la detección de disfunción ventricular izquierda en pacientes de alto riesgo en atención primaria / Validity and usefulness of B-type natriuretic peptide (BNP) for early detection of left ventricular dysfunction in high-risk patients in Primary Care

Objetivo: Determinar la validez y utilidad del BNP en atención primaria para detectar la disfunción ventricular en pacientes asintomáticos con alto riesgo de insuficiencia cardiaca (IC). Diseño: Estudio descriptivo prospectivo y multicéntrico de validación de prueba diagnóstica. Emplazamiento: Consultas de atención primaria de 7 centros de salud de la comunidad de Madrid. Participantes: Muestra consecutiva de 204 pacientes asintomáticos con riesgo elevado de presentar IC (estadios A y B de la American Heart Association). Mediciones principales: Se recogieron datos de la anamnesis, exploración física, electrocardiograma, factores de riesgo de IC y tratamiento actual. Se determinó el BNP en sangre venosa en la propia consulta mediante Triage BNP Test® (Biosite®) realizándose en las siguientes 72h un ecocardiograma (prueba de referencia). Comparamos los niveles de BNP según presencia o ausencia de disfunción ventricular, tipo (sistólica/diastólica) y grado. Se calcularon la sensibilidad, especificidad y los valores predictivos para el mejor punto de corte en la curva ROC. Resultados: Los valores de BNP fueron más altos (p<0,001) en pacientes con disfunción sistólica ventricular izquierda (DSVI). No se hallaron diferencias significativas para la disfunción diastólica. El mejor punto de corte para la detección de DSVI fue 71,00 pg/ml, siendo el área bajo la curva de 0,757 (IC 95%: 0,64-0,87). La sensibilidad fue del 75% (IC 95%: 50,66-99,34), especificidad 70,19% (62,81-77,57), valor predictivo positivo 20% (IC 95%: 9,05-30,95) y valor predictivo negativo 96,58% (IC 95%: 92,86-100), siendo la prevalencia de DSVI en esta población del 9,04%. Conclusiones: El BNP puede tener utilidad en el diagnóstico precoz de DSVI en pacientes de alto riesgo de IC en consultas de atención primaria debido a su alto VPN (> 96%)(AU)

Objective: The aim of this study was to determine the accuracy of BNP test for early diagnosis of left ventricular dysfunction in patients at high-risk for heart failure. Design: Cross-sectional descriptive study. Setting: 7 Primary Care Centres in Madrid (Spain). Participants: A consecutive sample of 204 consecutive asymptomatic patients with high risk for heart failure (Stages A-B, AHA/ACC Classification). Main measurements: BNP plasma levels were measured in the clinical setting using Triage BNP Test® (Biosite®) and an echocardiography was performed in the following 3 days in a single hospital unit as a reference standard. Plasma BNP levels were compared depending on the presence/absence of left ventricular dysfunction (LVD), type and severity degree. Sensitivity, specificity, positive and negative predictive values, and Área under the receiver operating characteristic curve (ROC) for BNP assay were calculated. Results: BNP values were significantly higher (P<.001) in patients with left ventricular systolic dysfunction (LVSD). No significant differences were found for diastolic dysfunction. The best cut-off value to discriminate the patients with LVSD was 71.00 pg/ml, with an Área under the ROC curve of 0.757 (95% CI 0.64-0.87). Sensitivity for LVD diagnosis was 75% (95% CI 50.66-99.34), specificity 70.19% (95% CI 62.81-77.57), positive predictive value (PPV) 20% (95% CI 9.05-30.95), and negative predictive value (NPV) 96.58% (95% CI 92.86-100), with LVSD prevalence of 9.04% in this population. Conclusions: BNP determinations are of value in diagnosing LVSD in a primary care setting, with similar sensitivities and specificities. Due to the high NPV is useful to rule-out patients for echocardiography(AU)

Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli.

To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant human urodilatin, a hormone for the treatment of acute decompensated heart failure. The fusion protein, which was overexpressed mainly as inclusion bodies in Escherichia coli, constituted about 25% of the total cell proteins. After purification by Ni-sepharose affinity chromatography and renaturation in refolding buffer, the fusion protein was cleaved with SUMO protease 1. Urodilatin was separated from the fusion partner by the subtractive chromatography using Ni-sepharose once again, and then further purified with reverse-phase high performance liquid chromatography. In vitro activity assay demonstrated that the recombinant urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.77+/-0.53 microg/ml, which was similar to that of the synthetic urodilatin standard. The expression strategy presented in this study allows convenient high yield and easy purification of small recombinant peptides with native sequences.

Expression, purification and characterization of human urodilatin in E. coli.

Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.

Multiple natriuretic peptides coexist in the most primitive extant ray-finned fish, bichir Polypterus endlicheri.

Natriuretic peptides (NPs) have diversified from a single NP in cyclostomes and elasmobranchs to multiple NPs in ray-finned fishes where ANP, BNP, VNP, and/or up to four CNPs (CNP-1, 2, 3, and/or 4) have been identified. To trace the evolutionary diversification of NPs in fishes, we analyzed the bichir (Polypterus endlicheri), believed to be the most primitive extant ray-finned fish, for the presence of any NPs by a PCR-based method using primers that amplify all NP cDNAs identified to date. We have cloned cDNAs encoding ANP, BNP, VNP from the heart and three CNPs (CNP-1, 3, and 4) from the brain. An extensive search for CNP-2 from the brain was not successful. The C-terminus of bichir ANP presented an amidation signal as in ray-finned fish ANP. The bichir BNP mRNA had AUUUA repeats in the 3'-untranslated region as observed in all BNP cDNAs of vertebrates. The bichir VNP had a long C-terminal 'tail' sequence extending from the intramolecular ring as does teleost VNP. The three bichir CNPs are structurally similar to each teleost counterpart and are grouped after molecular phylogenetic analyses. ANP was most abundantly expressed in the atrium, BNP in the ventricle, and VNP was expressed in both atrium and ventricle. The three CNPs are most abundantly expressed in the brain, and CNP-4 transcripts were found in small amounts in the ventricle and kidney. Taken together, it is clear that all major NPs exist prior to the whole genome duplication that occurred in the teleost lineage. Furthermore, this is the first observation that CNP-3, ANP, BNP, and VNP, whose genes are colocalized in the same chromosome, coexist in a single fish species including teleosts, thereby confirming that CNP-3 is not an ortholog of VNP, and that ANP, BNP, and VNP genes were generated by tandem duplication from the CNP-3 gene.

Overexpression and purification of recombinant atrial natriuretic peptide using hybrid fusion protein REF-ANP in Escherichia coli.

Atrial natriuretic peptide (ANP), a small peptide consisting of 28 amino acids, has been applied in clinical treatment for heart failure, but it can encounter proteolytic degradation during its expression in host cells. Therefore, it is usually reported that ANP was expressed as a part of fusion protein. The aim of our study was to use an overexpression system to express the fusion protein REF-ANP and to optimize a purification method. First, Escherichia coli DH5alpha was transformed with constructed expression vector containing two tandem copies of ref-anp gene and the fusion protein REF-ANP was overexpressed in shaking flask culture. Subsequently, the inclusion bodies were purified with reverse phase chromatography and pooled fractions were lyophilized. After this step, REF-ANP can be solubilized under native conditions without urea. After cleavage reaction, the sample was subjected to size exclusion chromatography and then rANP was polished with reverse phase chromatography. The final purity of rANP was more than 98% and the recovery of rANP per liter of shaking flask culture was more than 3mg. Such methods as mass spectrometry, capillary isoelectrofocusing analysis, and N-terminal amino acid sequence were used to identify rANP. The capillary isoelectrofocusing analysis showed that the pI of ANP was about pH 9.7. In this study, an efficient refolding and purification process should make scaling-up procedures easier and more successful than earlier reports. Moreover, it is possible that the refolding and purification method along with the overexpression system described in this article may offer new ideas on optimizing expression and purification of other kinds of short peptides.

Atrial natriuretic hormone, vessel dilator, long-acting natriuretic hormone, and kaliuretic hormone decrease the circulating concentrations of CRH, corticotropin, and cortisol.

The present investigation was designed to determine whether atrial natriuretic peptides consisting of amino acids 1-30 (i.e. long-acting natriuretic hormone), 31-67 (vessel dilator), 79-98 (kaliuretic hormone), and 99-126 (atrial natriuretic hormone) of the 126 amino acid atrial natriuretic hormone prohormone decrease CRH, ACTH, and/or cortisol in healthy humans (n = 30). Vessel dilator, kaliuretic hormone, long-acting natriuretic hormone, and atrial natriuretic hormone decreased the circulating concentration of CRH 84%, 74%, 67%, and 62% (P < 0.001 for each), respectively, when infused at 100 ng/kg body weight.min for 60 min. Vessel dilator, kaliuretic hormone, long-acting natriuretic hormone, and atrial natriuretic hormone decreased circulating ACTH concentrations 58%, 80%, 81%, and 70% (P < 0.001) and the circulating concentration of cortisol 73%, 72%, 73%, and 67% (P < 0.001), respectively. The decreases in CRH, ACTH, and cortisol lasted 11/2 to 3 h after cessation of the respective atrial natriuretic peptide infusions. These data, along with the knowledge that cortisol upregulates atrial natriuretic peptides' gene expression and CRH and ACTH stimulate atrial natriuretic peptides' release, suggest that these four atrial natriuretic peptides may be part of an intricate feedback system to help regulate cortisol concentrations via their ability to decrease the circulating concentration of CRH which, in turn, results in a decrease in ACTH and cortisol.

Chamber formation and morphogenesis in the developing mammalian heart.

In this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and beta-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of "primary" myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium.

Structure elucidation of a purple peptide found during the purification of a recombinant protein from Escherichia coli.

A purple substance (4) partially co-purified with a recombinant human B-type natriuretic peptide (hBNP), following an E. coli fermentation. The structure of the compound was elucidated by NMR, electrospray and FAB mass spectrometry. The chromophore is a 1,4-naphthoquinone condensed with the N-terminal cysteine of a heptapeptide by its NH2- and SH-groups to form a dihydro-thiazine ring. The peptide sequence was determined as Cys-Lys-Val-Leu-Arg-Arg-His by mass spectrometric techniques. CID and data base matching identified it as the C-terminus of the 32-amino-acid recombinant peptide hBNP. This modification of an N-terminal Cys may be a more general phenomenon with implications for the production of heterologous proteins by microorganisms.

Equine cardiodilatin/ atrial natriuretic peptide. Primary structure and immunohistochemical localization in auricular cardiocytes.

Cardiodilatin (CDD)/atrial natriuretic peptide (ANP) is a 28-amino acid peptide hormone known to be synthesized in the heart of a large number of different vertebrates. It plays an important role in the regulation of blood pressure and natriuresis/diuresis. Since the cardiovascular system of the horse has to meet the highest requirements concerning its physiological performance, we intended to characterize the cardiodilatin/atrial natriuretic peptide system of this species. By means of immunohistochemistry and immunoelectron microscopy, we precisely identified auricular cardiocytes as the loci of CDD/ANP synthesis. Using aortic smooth muscle relaxation assay and CDD/ANP-ELISA, we succeeded in isolating the biologically active prohormone. We subsequently cloned the equine cDNA of the CDD/ANP precursor protein and deduced its primary sequence. The entire precursor protein is in good agreement with the CDD/ANP prohormones of other mammals. The deduced theoretical average Mr of equine CDD/ANP-1-126 is 13,764, corresponding to the molecular weight of purified peptide determined by ESI-MS. Our findings suggest that equine CDD/ANP is produced in auricular cardiocytes and the predominant storage form of CDD/ANP in the auricle is the prohormone CDD/ANP-1-126.

Extraction of human alpha atrial natriuretic peptide and its physiological validation.

A very sensitive and specific radioimmunoassay for human alpha atrial natriuretic peptide (hANP) and a novel extraction method for hANP, have been developed. Antiserum to hANP showed no cross-reactivity with related analogues (e.g., brain natriuretic peptide). The radioimmunoassay can detect 1.2 fmol ANP/assay tube. Using a commercially available tracer, the antiserum binds 0.7 fmol of radioligand at a final dilution of 1:96,000. Production of ANP tracer, using 125I, Iodogen and reversed-phase HPLC separation, produces two products. The first has identical properties to the commercial reagent resulting in an identical antibody titre. The second, however, is more reactive with the antiserum which can be employed at a final dilution of 1:192K. These products represent oxidised and reduced peptides, respectively, inferring that the commercial tracer is oxidised. The recovery of synthetic hANP from plasma over the range of 0-1000 ng/l through Sep-Pak C18 cartridges, using an extraction method of acetic acid-acetonitrile (4:96) was 89%. Inter- and intra-assay coefficients of variation were 9.5% and 8.2%, respectively. The radioimmunoassay was validated in man by measuring plasma ANP (ng/l) following change of posture and exercise in normal man. Plasma ANP rose from 13.2 (1.0; S.D.) to 20.1 (1.6) from supine to sitting position. Plasma ANP increased to 20.1 (1.6) at rest (sitting) to 34 (2.7) ng/l at peak of exercise, but decreased from 31.2 (2.5) to 21.4 (0.1) ng/l at 3 and 6 min after exercise, respectively. These results confirm that the assay is capable of differentiating changes of concentrations within the physiological range.

Local synthesis of natriuretic peptides in the eel intestine.

The intestine is a major osmoregulatory organ in euryhaline fishes which allows them to survive in the sea, and natriuretic peptides have been implicated in regulation of transmural transport. Atrial (ANP) and ventricular natriuretic peptide (VNP) were identified in eel intestine. Elution profiles of ANP and VNP from high-performance liquid chromatography (HPLC) were determined by radioimmunoassay using highly specific antisera. Elution times of immunoreactive ANP and VNP in HPLC were identical to those of authentic peptide standards and were consistent with the relative molecular masses of these peptides. Tissue localization of ANP and VNP was accomplished by fluorescence immunohistochemistry. Immunoreactive cells were observed in the epithelium of anterior, middle, and posterior regions of intestine. Reverse transcription of mRNA isolated from intestine and subsequent polymerase chain reaction amplification yielded appropriate-size products consistent with ANP and VNP expression. Together, these data show that natriuretic peptides are synthesized locally in eel intestine, rather than trapped from the circulation.

A new natriuretic peptide isolated from cardiac atria of trout, Oncorhynchus mykiss.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are two cardiac natriuretic peptides (NPs) found in tetrapods from amphibians to mammals, whereas ANP and ventricular NP (VNP) have been identified in eel hearts. Because VNP has also been found in the rainbow trout ventricle, we attempted to isolate NP from trout cardiac atria in order to determine whether ANP and VNP are common cardiac NPs in teleosts. In the present experiments, we isolated VNP and a novel atrial NP consisting of 29 amino acid residues from the atria. This new trout NP exhibited similar sequence identity to mammalian ANP and BNP (50-60%). Its homology to eel ANP was low (52%) compared with high homology of trout and eel VNP (78%). Based on yield, the content of this new NP in trout atria may be even smaller than that of VNP. The new trout atrial NP exhibited low relaxant activity in the chick rectum (only 1/10 of that of trout VNP), and extremely low vasorelaxant activity in the rat aortic strip (only 1/400 of that of human ANP). However, the new trout NP was equipotent with trout VNP and human ANP in relaxing trout epibranchial artery. Based on the sequence similarity with other NPs and on atrial content, the new NP isolated from trout atria cannot yet be assigned to a known member of the NP family.

Development of immunoenzymatic reactions of peptides using recombinant hybrid proteins.

A recombinant hybrid protein comprising the bacteriophage fr coat protein, a short linker, and atrial natriuretic factor (ANF) as well as native and recombinant phage coat proteins, ANF, and variants of the hybrid recombinant protein were used in the development of various immunological reactions including immunization, preparation of affinity columns, purification of antibodies, synthesis of conjugates, and immunoenzyme assay of ANF and the recombinant protein. The hybrid protein is effective in competitive assay of ANF and other constructs that include the phage protein.

Synthesis of recombinant atrial natriuretic peptide (rANP) using hybrid fusion protein-phage fr coat/ANP (CP/ANP).

Recombinant atrial natriuretic peptide (rANP) was expressed in and isolated from E. coli. rANP was purified using HPLC. Amino acid analysis, partial sequencing, and molecular mass were determined. Fused protein was used to rise polyclonal antibodies and to develop of immunoenzymatic assays of rANP and CP/ANP. Experiments were designed to study rANP effects on isolated rabbit aortic strips and to examine hypotensive, diuretic, and natriuretic activity, as well as renal creatinine clearance, in an in vivo rat model. Identity of recombinant and commercial ANP has been confirmed. Physiological activity of CP/ANP has allowed the investigators to predict the conformation of CP/ANP, pro-ANP processing, and the method by which fusion protein interacts with ANP receptors.

Natriuretic peptide system in the rat submaxillary gland.

Natriuretic peptides and their receptors were characterized in rat submaxillary glands (SGs). Reverse phase-high performance liquid chromatography (HPLC) of rat SGs extracts revealed the presence of the 28-amino-acid (AA) circulating peptide ANP (Ser99-Tyr126) and the 126-AA prohormone (Asn1-Tyr126). The presence of ANP prohormone indicated that SGs are a site of ANP synthesis. Indeed, ANP mRNAs were demonstrated. ANP mRNA was 10 times lower than in the lung and only about 7 times lower than in the hypothalamus. ANP content in SG was determined as 30 +/- 8 ng/mg of protein (n = 7). In addition the presence of another member of the natriuretic peptide family, C-type natriuretic peptide (CNP), was found in SG. The CNP level of 293 +/- 38 pg/mg protein was significantly higher than in the lungs (44 +/- 6 pg/mg protein, P < 0.001, n = 5), but about 15 times lower than in hypothalamus (4.5 +/- 0.6 ng/mg protein, P < 0.001, n = 6). Both guanylyl cyclase and clearance receptors were expressed in SG. The presence of natriuretic peptide transcripts and their receptors suggests a role in rat SG functions.

Immunohistochemical identification and effects of atrial natriuretic peptide, pancreastatin, leucine-enkephalin, and galanin in the porcine pancreas.

This study demonstrates the presence and distribution of atrial natriuretic peptide (ANP) pancreastatin (PST), leucineenkephalin (Leu-ENK), galanin (GAL), and insulin in the pig pancreas. The effects of PST, ANP, Leu-ENK, and GAL on protein and amylase secretion were also investigated to determine their functional role in the control of pancreatic secretion. PST-immunoreactive cells were observed in the islet of Langerhans and in the wall of the ducts. Leu-ENK-immunopositive cells were observed in both the endo-and exocrine pancreas. It is colocalized with insulin in the islet of Langerhans. ANP immunoreactivity was discernible in nerve fibers and cells of the exocrine pancreas. GAL-immunopositive cells were observed in close association with insulin-positive cells in the islets of Langerhans and in the exocrine pancreas. Stimulation of isolated pancreatic segments with either ANP or Leu-ENK resulted in increased protein secretion and amylase output. The Leu-ENK-evoked amylase secretion was antagonized by naloxone. Pancreastatin was effective at all concentrations, but low concentration had more marked secretory effects whereas GAL failed to evoke any significant increases in either protein or amylase secretion. The results of the study have demonstrated a close association of peptidergic fibers with the secretory cells of the pancreas. The nerve fibers can release peptides that in turn can stimulate protein and amylase secretion.

Purification by immobilized metal affinity chromatography of human atrial natriuretic peptide expressed in a novel thioredoxin fusion protein.

A fusion protein that contains human atrial natriuretic peptide (ANP) at its carboxy terminus has been genetically engineered with the objective of being able to produce the peptide in a process with a relatively simple purification procedure. The fusion protein also includes a (His)6 metal affinity binding site at the amino terminus, followed by Escherichia coli thioredoxin, a factor Xa protease recognition site, and ANP. With induction of the tac promoter at 30 degrees C, the expression level of the fusion protein was high (10% of total cell protein as measured by densitometry) and it was almost completely (92%) expressed as a soluble protein in the cytoplasm. A step gradient elution with imidazole of a column of Ni2+ chelated to iminodiacetic acid-agarose saturated with proteins in crude cell extract gave a very nearly pure fusion protein. After digestion of the purified fusion protein with factor Xa protease, ANP of exactly the correct size (to within 2 Da) was observed by coupled HPLC/mass spectrometry.

Temporal (circadian) and functional relationship between atrial natriuretic peptides and blood pressure.

Long-acting natriuretic peptide, vessel dilator, and atrial natriuretic factor consisting of amino acids (a.a.) 1 to 30, 31 to 67, and 99 to 126 of the 126-a.a. atrial natriuretic factor (ANF) prohormone, respectively, circulate in humans and have potent vasodilatory properties. To determine if these atrial natriuretic peptides are directly related to blood pressure in clinically healthy normotensive humans, we obtained 24-h profiles of vessel dilator, long-acting natriuretic peptide, ANF, and blood pressure in 10 men in 1988 and 11 men in 1993 (seven men were studied twice) to compare circulating concentrations of atrial natriuretic peptides with naturally occurring changes in blood pressure. Overall, vessel dilator, long-acting natriuretic peptide, and ANF each had significant (p<0.001) circadian rhythms, with peak concentrations late during sleep (at 04:00 h) being nearly twice their concentrations in the afternoon and evening. This high-amplitude circadian change allowed for the refinement of normal limits for ANF peptides by computing 3-hourly tolerance intervals (chronodesms) against which to compare time-specified single samples for normality. Systolic, diastolic, and mean arterial blood pressure also had significant circadian rhythms (p<0.001) with peaks and troughs that were exactly opposite those of the ANF peptides. In addition to this inverse temporal relationship, there was a significant inverse correlation between absolute values for blood pressure and each ANF peptide (p<0.001), implying a functional relationship. These data suggest that in addition to other well-established neurochemical factors, the ANF peptides (vessel dilator, long-acting natriuretic peptide, and ANF) are important for the maintenance of blood pressure and modulation of its circadian rhythm.