Biomedical subjects

J K Gierse

Publications and source records attributed to J K Gierse.

At least 19 recordsLinked to original sources

PubMed2001

A three-step kinetic mechanism for selective inhibition of cyclo-oxygenase-2 by diarylheterocyclic inhibitors.

Cyclo-oxygenase (COX) enzymes are the targets for non-steroidal anti-inflammatory drugs (NSAIDs). These drugs demonstrate a variety of inhibitory mechanisms, which include simple competitive, as well as slow binding and irreversible inhibition. In general, most NSAIDs inhibit COX-1 and -2 by similar mechanisms. A unique class of diarylheterocyclic inhibitors has been developed that is highly selective for COX-2 by virtue of distinct inhibitory mechanisms for each isoenzyme. Several of these inhibitors, with varying selectivity, have been utilized to probe the mechanisms of COX inhibition. Results from analysis of both steady-state and time-dependent inhibition were compared. A generalized mechanism for inhibition, consisting of three sequential reversible steps, can account for the various types of kinetic behaviour observed with these inhibitors.

Animals↗

PubMed2000

Structural insights into the stereochemistry of the cyclooxygenase reaction.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

Animals↗

PubMed2000

Spatial requirements for 15-(R)-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2. Why acetylated COX-1 does not synthesize 15-(R)-hete.

The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (15-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11- and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-1 equivalents resulted in abrogation of 11- and 15-HETE production after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the omega-end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site-directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel. Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.

Acetylation↗

PubMed2000

Structure-activity relationship studies on 1-[2-(4-Phenylphenoxy)ethyl]pyrrolidine (SC-22716), a potent inhibitor of leukotriene A(4) (LTA(4)) hydrolase.

Leukotriene B(4) (LTB(4)) is a pro-inflammatory mediator that has been implicated in the pathogenesis of a number of diseases including inflammatory bowel disease (IBD) and psoriasis. Since the action of LTA(4) hydrolase is the rate-limiting step for LTB(4) production, this enzyme represents an attractive pharmacological target for the suppression of LTB(4) production. From an in-house screening program, SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) was identified as a potent inhibitor of LTA(4) hydrolase. Structure-activity relationship (SAR) studies around this structural class resulted in the identification of a number of novel, potent inhibitors of LTA(4) hydrolase, several of which demonstrated good oral activity in a mouse ex vivo whole blood assay.

Administration, Oral↗

PubMed1999

Kinetic basis for selective inhibition of cyclo-oxygenases.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the formation of prostaglandins by cyclo-oxygenases (COX). The discovery of a second COX isoform (COX-2) associated with inflammation led to agents that selectively inhibit COX-2, e.g. celecoxib. We evaluated the kinetics of inhibition of celecoxib and several NSAIDs. Celecoxib displays classic competitive kinetics on COX-1 (Ki=10-16 microM). An initial competitive interaction with COX-2 can also be discerned with celecoxib (Ki=11-15 microM), followed by a time-dependent interaction leading to potent inhibition, characterized as inactivation (Kinact=0.03-0.5 s-1). Half-maximal inhibition (IC50) using end-point assays reflects the competitive component on COX-1 (IC50=4-19 microM) and the inactivation component on COX-2 (IC50=0.003-0.006 microM). NSAIDs exhibit four distinct modes of COX inhibition based on kinetic behaviour: (1) competitive, e.g. ibuprofen; (2) weak binding, time-dependent, e.g. naproxen, oxicams; (3) tight binding, time-dependent, e.g. indomethacin; (4) covalent, e.g. aspirin. In addition, most NSAIDs display different kinetic behaviour for each isoform. Weakly binding inhibitors show variable behaviour in enzyme assays, with apparent inhibitory activity being markedly influenced by experimental conditions; determination of kinetic constants with this class is unreliable and IC50 values are strongly dependent on assay conditions. Although IC50 determinations are useful for structure/activity analyses, the complex and distinct mechanisms of enzyme inhibition of each COX isoform by the NSAIDs renders comparison of inhibitory activity on COX-1 and COX-2 using IC50 ratios of questionable validity.

Animals↗

PubMed1997

Mutational analysis of the role of the distal histidine and glutamine residues of prostaglandin-endoperoxide synthase-2 in peroxidase catalysis, hydroperoxide reduction, and cyclooxygenase activation.

Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wtPGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to approximately 60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS.

Animals↗

PubMed1997

Nitric oxide trapping of the tyrosyl radical of prostaglandin H synthase-2 leads to tyrosine iminoxyl radical and nitrotyrosine formation.

The determination of protein nitrotyrosine content has become a frequently used technique for the detection of oxidative tissue damage. Protein nitration has been suggested to be a final product of the production of highly reactive nitrogen oxide intermediates (e. g. peroxynitrite) formed in reactions between nitric oxide (NO.) and oxygen-derived species such as superoxide. The enzyme prostaglandin H synthase-2 (PHS-2) forms one or more tyrosyl radicals during its enzymatic catalysis of prostaglandin formation. In the presence of the NO.-generator diethylamine nonoate, the electron spin resonance spectrum of the PHS-2-derived tyrosyl radical is replaced by the spectrum of another free radical containing a nitrogen atom. The magnitude of the nitrogen hyperfine coupling constant in the latter species unambiguously identifies it as an iminoxyl radical, which is likely formed by the oxidation of nitrosotyrosine, a stable product of the addition of NO. to tyrosyl radical. Addition of superoxide dismutase did not alter the spectra, indicating that peroxynitrite was not involved. Western blot analysis of PHS-2 after exposure to the NO.-generator revealed nitrotyrosine formation. The results provide a mechanism for nitric oxide-dependent tyrosine nitration that does not require formation of more highly reactive nitrogen oxide intermediates such as peroxynitrite or nitrogen dioxide.

Arachidonic Acid↗

PubMed1996

A single amino acid difference between cyclooxygenase-1 (COX-1) and -2 (COX-2) reverses the selectivity of COX-2 specific inhibitors.

Nonsteroidal anti-inflammatory drugs (NSAIDs) currently available for clinical use inhibit both COX-1 and COX-2. This suggests that clinically useful NSAIDs inhibit pro-inflammatory prostaglandins (PGs) derived from the activity of COX-2, as well as PGs in tissues like the stomach and kidney (via COX-1). A new class of compounds has recently been developed (SC-58125) that have a high degree of selectivity for the inducible form of cyxlooxygenase (COX-2) over the constitutive form (COX-1). This unique class of compounds exhibit a time-dependent irreversible inhibition of COX-2, while reversibly inhibiting COX-1. The molecular basis of this selectivity was probed by site-directed mutagenesis of the active site of COX-2. The sequence differences in the active site were determined by amino acid replacement of the COX-2 sequences based on the known crystal structure of COX-1, which revealed a single amino acid difference in the active site (valine 509 to isoleucine) and a series of differences at the mouth of the active site. Mutants with the single amino acid substitution in the active site and a combination of three changes in the mouth of the active site were made in human COX-2, expressed in insect cells and purified. The single amino acid change of valine 509 to isoleucine confers selectivity of COX-2 inhibitors in the class of SC-58125 and others of the same class (SC-236, NS-398), while commonly used NSAIDs such as indomethacin showed no change in selectivity. Substitutions of COX-1 sequences in COX-2 at the mouth of the active site of COX-2 did not change the selectivity of SC-58125. This indicates that the single amino acid substitution of isoleucine at position 509 for a valine is sufficient to confer COX-2 selectivity in this example of a diaryl-heterocycle COX inhibitor.

Amino Acid Sequence↗

PubMed1995

Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase.

The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.

Animals↗

PubMed1994

The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity.

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are "better" substrates. This is compatible with a biological role for the peptidase activity of the enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.

Amino Acid Sequence↗

PubMed1993

High-level expression and purification of human leukotriene A4 hydrolase from insect cells infected with a baculovirus vector.

Leukotrienes constitute a group of bioactive compounds derived from arachidonic acid which play important roles in immediate hypersensitivity and inflammation. Leukotriene A4 hydrolase (LTA4H) is an epoxide hydrolase, catalyzing the hydration of LTA4 to LTB4, and also acts an aminopeptidase, with the ability to cleave amides of p-nitroaniline. The cDNA for LTA4H was cloned using oligonucleotide-directed amplification of the cDNA sequence by polymerase chain reaction and by oligonucleotide-based screening of a bacteriophage lambda gt11 cDNA library derived from human placental tissue. High levels of biologically active LTA4H were expressed in cultured Spodoptera frugiperda insect cells infected with a baculovirus expression vector containing the LTA4H cDNA. Expression levels were approximately 100 mg per liter of cell-free culture media. LTA4H was recovered from the medium and purified to > 95% purity by ion-exchange and gel-filtration chromatography, with an overall yield of 76%. LTA4H produced by insect cells exhibits both hydrolase and aminopeptidase activities and has kinetic properties similar to those reported for enzyme isolated from human lung. Two major isoforms, with pI's of 5.3 and 5.1, were isolated by preparative chromatofocusing chromatography. NH2-terminal sequence analysis revealed that the two different by an NH2-terminal blocking group. Electrospray ionization mass spectrometry indicates that the two isoforms differ by a molecular mass of 42, indicating that the blocking group is an acetyl group.

Acetylation↗

PubMed1992

Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli.

In Escherichia coli high-level production of some heterologous proteins (specifically, human prorenin, renin, and bovine insulin-like growth factor 2) resulted in the induction of two new E. coli heat shock proteins, both of which have molecular masses of 16 kDa and are tightly associated with inclusion bodies formed during heterologous protein production. We named these inclusion body-associated proteins IbpA and IbpB. The coding sequences for IbpA and IbpB were identified and isolated from the Kohara E. coli gene bank. The genes for these proteins (ibpA and ibpB) are located at 82.5 min on the chromosome. Nucleotide sequencing of the two genes revealed that they are transcribed in the same direction and are separated by 110 bp. Putative Shine-Dalgarno sequences are located upstream from the initiation codons of both genes. A putative heat shock promoter is located upstream from ibpA, and a putative transcription terminator is located downstream from ibpB. A temperature upshift experiment in which we used a wild-type E. coli strain and an isogenic rpoH mutant strain indicated that a sigma 32-containing RNA polymerase is involved in the regulation of expression of these genes. There is 57.5% identity between the genes at the nucleotide level and 52.2% identity at the amino acid level. A search of the protein data bases showed that both of these 16-kDa proteins exhibit low levels of homology to low-molecular-weight heat shock proteins from eukaryotic species.

Amino Acid Sequence↗

PubMed1991

Production of bovine insulin-like growth factor 2 (bIGF2) in Escherichia coli.

Bovine insulin-like growth factor 2 (bIGF2) was produced in inclusion bodies in the cytoplasm of Escherichia coli and accumulated at high levels: 20-25% of total Coomassie-stained bacterial protein. The level of accumulation of bIGF2 was affected by the choice of codons in the 5' end of the coding sequence and by a rpoH mutation in the host cells. Purified recombinant bIGF2 had the native N terminus and the same mitogenic activity as that of bIGF2 purified from bovine serum.

Amino Acid Sequence↗

PubMed1990

Substrate specificity of recombinant human renal renin: effect of histidine in the P2 subsite on pH dependence.

Steady-state kinetic analysis of human renin demonstrates the histidine proximal to the substrate scissile peptide bond contributes to the unique specificity and pH dependence of this aspartyl protease. Recombinant human renal renin purified from mammalian cell culture appears to be indistinguishable from renin isolated from human kidney with respect to specific activity (1000 Goldblatt units/mg). Recombinant renin contains carbohydrate covalently attached to asparagines at positions 5 and 75 (renin numbering) and disulfide linkages at Cys-51/Cys-58, Cys-217/Cys-221, and Cys-259/Cys-296. Renin pH dependence was evaluated between pH 4.0 and 8.0 by using a synthetic substrate identical with the amino terminus of porcine angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu*Leu-Val-Tyr-Ser, where the asterisk indicates the scissile peptide bond and the proximal histidine is in italics) and an analogous tetradecapeptide where the proximal histidine was substituted with glutamine. Comparison of the pH profiles shows the catalytic efficiency (V/Km) and maximal velocity (V) of renin are greater above pH 6.5 with the substrate containing histidine proximal to the scissile peptide bond, but below pH 5.0 these parameters are greater with the glutamine substrate analogue. Solvent isotope effects show that proton transfer contributes to the rate-limiting step in catalysis with both substrates and that the proximal histidine does not serve as a base in the catalytic mechanism. Molecular modeling indicates the substrate histidine could hydrogen bond to Asp-226 of the enzyme (renin numbering), thus perturbing the ionization of the catalytic aspartyl groups (Asp-38 and Asp-226).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Alternative mechanisms for atriopeptin prohormone processing by isolated perfused rat hearts.

The isolated perfused rat heart releases atriopeptin-28 [AP28 (ANF99-126)], whereas the storage form of AP in the heart is the intact prohormone AP126 (ANF1-126). Right atrial stretch or phenylephrine (5 x 10(-5) M) stimulated the release of AP28. The processing of the prohormone during stretch was inhibited by infusion of the protease inhibitor aprotinin, resulting in the appearance of intact AP126 in the cardiac effluent. Other protease inhibitors including p-aminobenzamidine and soybean trypsin inhibitor did not alter prohormone processing by the isolated heart subjected to stretch. In contrast, aprotinin did not block the prohormone processing induced by phenylephrine. Ca+(+)-free medium markedly inhibited prohormone processing during stretch without a significant effect on AP release, whereas phenylephrine-stimulated AP release was completely suppressed by Ca+(+)-free medium. Exogenous AP126 could be cleaved by isolated rat hearts perfused either with Krebs-Henseleit solution or with Ca+(+)-free medium. However, amino acid sequence analysis revealed that the prohormone cleavage in Ca+(+)-free medium occurred at sites other than between Arg98 and Ser99 and that the resultant low molecular weight APs were not AP28. These findings suggest: 1) the characteristics of the enzyme(s) involved in the processing of AP prohormone in isolated perfused rat hearts are different from the described properties of purified enzymes; 2) in isolated perfused rat hearts the specific AP processing enzyme is Ca++ dependent, whereas nonspecific cleavage does not necessarily require Ca++ and 3) two independent AP processing pathways differentially activated by mechanical (stretch) and pharmacologic (alpha 1-adrenergic agonist) stimuli exist.

Animals↗

PubMed1989

Preliminary crystallographic study of glycosylated recombinant human renin.

Single crystals of glycosylated recombinant human renin have been obtained using the hanging-drop vapor diffusion method with polyethylene glycol and sodium chloride as coprecipitants. The crystals belong to the cubic space group P2(1)3 with a = 143.0 A and contain two molecules of renin in the asymmetric unit. A self-rotation function study using 5.5 A data shows the orientation of a non-crystallographic 2-fold axis relating these two monomers.

Glycosylation↗

PubMed1989

Expression, purification, and in vivo activity of atrial natriuretic factor prohormone produced in Escherichia coli.

Atrial muscles of the heart are known to produce polypeptide hormones called atrial natriuretic factors (ANF) which have potent diuretic and hypotensive action. These hormones are synthesized as a larger protein precursor called pro atrial natriuretic factor or proANF which contains the biologically active ANF sequences at its C-terminus. Rat proANF (representing amino acids -1 to 128 of the coding sequence) was expressed in a soluble form in Escherichia coli. A simple purification procedure was developed which consists of boiling E. coli cell extracts in 1 M acetic acid and subjecting the supernatant to reversed-phase HPLC. The effect of intravenous administration of the purified recombinant proANF on mean arterial blood pressure was examined. The displacement dose-response curves obtained demonstrated that proANF exhibits similar, albeit less potent, physiological activity than ANF.

Amino Acid Sequence↗

PubMed1988

Manipulation of stretch-induced atriopeptin prohormone release and processing in the perfused rat heart.

Atriopeptin (AP) is stored as the prohormone AP-126 [atrial natriuretic factor-(1-26)] in atrial granules. Cultured atrial myocytes synthesize and release only prohormone into the medium. HPLC analysis of the coronary venous effluent of media from perfused rat hearts subjected to right atrial stretch indicated the presence of the C-terminal mature hormone AP-28 [atrial natriuretic factor-(99-126)] and little or no prohormone. Absence of calcium from the perfusion medium increased total AP release and surprisingly blocked the proteolytic cleavage of the prohormone. Similarly, addition of the proteolytic inhibitor aprotinin to the perfusion medium suppressed the processing of the endogenous AP-126 released by atrial stretch. Aprotinin would be restricted to the extracellular space, which is therefore implicated as the site of prohormone processing. This suggestion was validated by the demonstration that the perfused rat heart could readily cleave exogenous prohormone to mature hormone, a process blocked by aprotinin. Hypothetically, the stimulus-release-processing event initiated by atrial stretch may require the concerted action of the synthetic cell (i.e., atrial myocyte) and a processing cell or site (e.g., the adjacent atrial mesenchymal cell) for the production of the mature AP-28, which is the circulating molecular form of this endocrine system.

Animals↗