Introduction

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More than half of the known peptides functioning as intercellular messengers require carboxy terminal -amidation for receptor recognition and biological activity (Eipper et al., 1992a). -Amidation is catalyzed in a two-step process by the sequential actions of peptidylglycine--hydroxylating monooxygenase (PHM; EC 1.14.17.3) and peptidyl--hydroxyglycine -amidating lyase (PAL; EC 4.3.2.5). PHM and PAL are typically expressed together as a large bifunctional enzyme, peptidylglycine--amidating monooxygenase (PAM; EC 1.14.17.3). PHM is rate limiting in -amidation and can control the overall production of -amidated peptides (Mains et al., 1991; Eipper et al., 1992a). Recent evidence that the expression (Ouafik et al., 1990; Meskini et al., 1997) and activity (Mueller et al., 1993; Prohaska et al., 1997) of PHM are both subject to regulation indicates that PHM is an important control point for the bioactivation of peptide messengers.

Bifunctional PAM is targeted to secretory granules in peptidergic cells, where it can be anchored to membrane by its membrane spanning domain. Alternatively, soluble forms of PHM, PAL, and bifunctional PAM are generated by alternate splicing of the single-copy PAM gene (Ouafik et al., 1992) and by differential post-translational endoproteolytic processing (Eipper et al., 1992b). Whereas PHM and PAL are normal constituents of the circulation (Eipper et al., 1985; Wand et al., 1985), presumably arising from active secretion of their soluble forms, neither the source nor the physiologic significance of blood-borne -amidating activity is understood.

To date, few tools have been developed for investigating the physiology of PHM in vivo; however, several classes of inhibitors of PHM, including olefinic compounds (Bradbury et al., 1990; Katopodis and May, 1990), N-substituted homocysteines (Erion et al., 1994), hydrazines (Merkler et al., 1995), and pyruvate-extended amino acid derivatives (Mounier et al., 1997), have been characterized in vitro. We examined the in vivo actions of the substrate analog 4-phenyl-3-butenoic acid (PBA; Fig. 1). PBA acts as a potent mechanism-based inhibitor that irreversibly inactivates PHM, reportedly through covalent modification (Bradbury et al., 1990). Whereas PHM inactivation has been demonstrated with purified PHM protein (Bradbury et al., 1990; Katopodis and May, 1990; Katopodis et al., 1990; Bolkenius et al., 1997) and in cell culture (Bradbury et al., 1990; Oldham et al., 1992), the effect of PBA on PHM activity in living systems is not well characterized. A recent report by Ogonowski and coworkers (Ogoniowski et al., 1997) shows that PBA significantly inhibits circulating PHM in rats, as noted earlier by Altarac et al. (1993). The novel results of our investigation demonstrate that in vivo PBA administration also effectively compromises tissue PHM activity in cardiac atrium, pituitary, and brain. Additionally, evidence is presented showing the importance of de novo protein synthesis as the mechanism for sustaining basal levels of PHM and for rapidly replenishing circulating PHM activity after its inactivation by PBA.

View larger version (5K): [in this window] [in a new window]   Fig. 1.   Structure of PBA (styrylacetic acid).

	Materials and Methods

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Animals, Treatments, and Sample Collection. Mature male rats (Charles River Laboratories, Wilmington, MA) were housed at 22°C under a 12-h daily light cycle and received food and water ad libitum. PBA (Aldrich Chemical Co., Inc., Milwaukee, WI) was dissolved in ethanol, diluted with corn oil to a final concentration containing 15% ethanol, and administered s.c. in a volume of 1 ml. Cycloheximide (Sigma Chemical Co., St. Louis, MO) was dissolved in 0.9% NaCl and administered i.p. in two doses of 3.3 mg/kg each at 4 and 8 h before sample collection. This dose regimen for cycloheximide was previously shown to inhibit protein synthesis in the pituitary by 95% (Shull and Gorski, 1985). After sacrifice (CO₂ anesthesia and decapitation), blood was collected and allowed to clot for 30 min at room temperature, and serum was prepared by centrifugation and stored at 70°C. Atrium, anterior and neurointermediate (NIL) pituitary lobes, and hypothalamus were quickly dissected and either processed immediately or snap frozen on dry ice and stored at 70°C.

Tissue Processing and Sample Preparation. Serum was diluted 1:10 in 0.05 M 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.0), 10 mM mannitol containing protease inhibitors [leupeptin (2.0 µg/ml), benzamidine (16 µg/ml), lima bean trypsin inhibitor (10 µg/ml), and phenylmethylsulfonyl fluoride (15 µg/ml)]. Tissues were homogenized in the same buffer and then processed through three freeze-thaw cycles, followed by centrifugation at 400g for 10 min at 4°C. The resulting supernatants were separated into soluble and particulate (membrane-bound) fractions by ultracentrifugation (435,000g for 15 min at 4°C). Supernatants were collected, and pellets were resuspended in 0.02 M N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 7.0), 10 mM mannitol, protease inhibitors, and Triton X-100 (1%; Surfact-AmpsX-100, Pierce, Rockford, IL). For total tissue activity determinations, 1% Triton X-100 was included in the homogenization buffer, and the high-speed supernatant was assayed. Protein content was determined by the bicinchoninic acid assay (Pierce, Rockford, IL).

Enzyme Assays. PHM activity was measured under optimal conditions according to minor modifications of an established procedure (Perkins et al., 1990a,b). The assay measures PHM activity in a manner that is independent of the activity of PAL. Briefly, all samples (0.5 µl of serum or 0.5 or 1 µg of tissue protein) were assayed with 0.5 µM -N-acetyl-Tyr-Val-Gly and ¹²⁵I-labeled -N-acetyl-Tyr-Val-Gly in 0.15 M NaMES (pH 5.0) containing 1 µM CuSO₄, 1 mM ascorbate, and 100 µg/ml catalase in 40 µl. PAL activity was measured with ¹²⁵I-labeled and 0.5 µM unlabeled -N-acetyl-Tyr-Val--hydroxyglycine in 150 mM NaMES (pH 5.0), 0.05% Triton X-100 as described earlier (Eipper et al., 1991).

Kinetic Analysis of PHM. PHM activity was assayed in quadruplicate with ¹²⁵I-labeled -N-acetyl-Tyr-Val-Gly (30,000-40,000 cpm) in the presence of 0.5 to 75 µM unlabeled acetyl-Tyr-Val-Gly. Eadie-Hofstee plots were constructed with 95% confidence intervals. Analysis of covariance revealed that all treatment conditions exhibited homogeneity in regression coefficients; differences in x and y intercepts were considered significant when 95% confidence intervals for the regression lines did not overlap.

Statistical Analyses. Statistical differences between treatment group means were determined with the Scheffé comparison test after ANOVA (p < .05).

	Results

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In Vitro Experiments. Soluble and membrane-bound forms of bifunctional PAM were used to assess the effect of PBA on PHM and PAL activities in vitro (Fig. 2). PBA produced a dose-dependent inhibition of PHM activity without affecting the activity of PAL. PHM inactivation was nearly complete at 10 µM PBA for both soluble and membrane-bound forms of PHM. The action of PBA as a mechanism-based inhibitor was confirmed by preincubating PHM with PBA (10 µM) under conditions that did not support catalysis (absence of copper and ascorbate). PHM treated in this manner exhibited full enzymatic activity when separated from PBA and then placed under optimal assay conditions (not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Selective inhibition of PHM by PBA. Soluble and membrane-bound forms of bifunctional PAM were prepared from rat atrium and assayed for PHM and PAL activities in the presence of increasing concentrations of PBA.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of pretreatment with PBA on the kinetic properties of PHM. The Eadie-Hofstee plot shows results of analyses performed on PHM in detergent-soluble fractions of atrium preincubated under assay conditions in the presence or absence of PBA (3.2 µM, 15 min). After exposure to PBA, samples were diluted and assayed in quadruplicate (0.15 µg of protein) for PHM activity in the presence of increasing concentrations of peptide substrate. The final concentration of PBA in the kinetic analysis (0.08 µM) was below the threshold concentration for direct inhibition of PHM activity (see Fig. 2). The data are representative of two independent analyses; Km and Vmax values for data shown are 28 versus 23 µM and 786 versus 476 pmol · µg1 · h1 for control and PBA-treated samples, respectively.

In Vivo Experiments. Inhibition of serum PHM activity by in vivo PBA treatment was dose related and relatively short lived (Fig. 4). A single administration of 500 mg/kg PBA reduced circulating PHM activity to less than 5% of control values by 3 h. This effect was fully reversed by 24 h after injection.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Dose response and time course for inhibition of serum PHM activity by PBA. Top, groups of rats (n = 8) were treated with the doses of PBA indicated and samples collected 3 h later. Bottom, groups of rats (n = 8) were treated with either 500 mg/kg PBA or vehicle and samples collected at the times indicated. Serum PHM activities were determined by assay in quadruplicate. Columns represent group means ± S.E. *p  .05, **p  .01, ***p  .001, versus control values.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Dose-response and time course inhibition of NIL PHM activity by PBA. Soluble and membrane-bound PHM were prepared from tissue homogenates by ultracentrifugation according to the experimental protocol detailed in Materials and Methods. *p  .05, **p  .01, versus control values.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of chronic PBA administration on serum and atrial PHM activities. Groups of rats (n = 8) were treated with either a single or daily injections of 500 mg/kg PBA. Samples were collected 3 h after the single injection of PBA or 24 h after the last daily injection. Control rats received a single injection of vehicle, and samples were collected 3 h later. Tissue homogenates were fractionated by ultracentrifugation to prepare samples of soluble and membrane-bound PHM. Assays for PHM activity were performed in quadruplicate. Columns represent group means ± S.E. *p  .05, ***p  .001, versus control values.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Dose-response and time course inhibition of hypothalamic PHM activity by PBA. Soluble and membrane-bound PHM were prepared from tissue homogenates by ultracentrifugation according to the experimental protocol detailed in Materials and Methods. *p  .05, **p  .01, versus control values.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Effect of cycloheximide treatment on the recovery of serum PHM activity after the administration of PBA. Top, time course for the recovery of serum PHM activity in groups of rats (n = 8) treated with a single injection of 500 mg/kg PBA. Samples were collected at the times indicated; control samples were collected 2 h after the administration of vehicle. Bottom, groups of rats (n = 8-10) received either cycloheximide (Cyclo; two sequential doses of 3.3 mg/kg at 4 and 8 h before sample collection), PBA [500 mg/kg at either 2 h (PBA-2) or 10 h (PBA-10) before sample collection], or PBA (500 mg/kg at 10 h) plus cycloheximide (3.3 mg/kg at 4 and 8 h before sample collection; PBA-10 + Cyclo). Assays for PHM activity were performed in quadruplicate. Columns represent group means ± S.E. *p  .05 compared with PBA-10.

	Discussion

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Drugs that inhibit rate-limiting enzymes have important uses as experimental tools and therapeutic agents. The findings presented here demonstrate that PBA inhibits circulating and tissue PHM and thus offers a pharmacological approach for investigating the regulation and function of this rate-limiting enzyme. Although significant advances have been made in our understanding of PHM in neural and endocrine tissues, virtually nothing is known about the physiology of blood-borne PHM, the form most profoundly inhibited by PBA treatment. Thus, PBA may prove to be especially useful in defining the functions of circulating PHM, in addition to increasing our understanding of PHM in peptidergic tissues.

Although the functions of blood-borne PHM remain to be determined, levels of circulating PHM are significant (Eipper et al., 1985; Wand et al., 1985; Ogonowski et al., 1997), and, as shown here, its turnover appears to be rapid. Several -amidated peptides have been implicated in the control of vasomotion by their direct actions on blood vessels (Walker et al., 1991; Bell and McDermott, 1996; Kangawa et al., 1996). These peptides, however, are thought to be -amidated in situ before release. A precedent for the -amidation of an intercellular effector peptide after its release into blood is lacking. Thus, circulating PHM may have functions that are unrelated to peptide -amidation. These could include functions as a hydroxylase in the generation of novel nonpeptide products.

Likely sources of circulating PHM include endothelial cells and cardiac atrium. Very little is known about the physiology of endothelial cell PAM or the extent to which it may be secreted (Oldham et al., 1992). The expression of PAM in cardiac atrium far exceeds that in all other tissues (Eipper et al., 1992a,b). Because the amounts of -amidated peptide produced by the heart are small, the primary purpose for the expression of PHM in atrium may be for secretion (Miller et al., 1996; Shi et al., 1996).

Our findings that PBA inhibits PHM activity in experimental animals were anticipated based on the knowledge that PBA acts directly to irreversibly inactivate PHM protein in vitro and in cultured cells (Bradbury et al., 1990). As shown here, the in vivo actions of PBA are of relatively short duration, even for blood-borne PHM, where enzyme activity is initially inhibited by more than 95% (Fig. 3; Ogonowski et al., 1997). This finding points to several possibilities concerning the pharmacology of PBA and the physiological mechanisms that govern PHM expression and activity. The rapid recovery of PHM activity in vivo supports the conclusion that PBA is metabolized and/or cleared rapidly. Accordingly, it was necessary to use lipid-based depot administration of PBA because equivalent doses of water-based formulations resulted in little or no inhibition of PHM activity in vivo (our unpublished findings). The importance of de novo protein synthesis in maintaining PHM activity in blood is demonstrated by the ability of cycloheximide treatment to both reduce serum PHM activity and retard the recovery of PHM activity after PBA inactivation. Although levels of PHM protein were not measured directly, cycloheximide itself has no direct effect on the enzyme's activity in vitro. Thus, it is reasonable to conclude that loss of PHM protein underlies the cycloheximide-induced decline in enzyme activity in vivo. Furthermore, the finding that an extended, high-dose regimen of PBA (500 mg/kg daily for 10 days) failed to appreciably alter circulating or tissue PHM activity when measured 24 h after the last injection indicates that there was no accumulation of either the drug or its effect and that protein synthesis is fully capable of rapidly restoring physiological levels of PHM activity under these treatment conditions. Thus, the mechanisms for degrading PBA and for replenishing PHM activity appear to be robust. Whereas PHM inactivated by PBA in vitro cannot be reactivated, the possibility that this transformation occurs in vivo and thus contributes to the observations reported cannot be ruled out.

No overt behavioral responses were observed after PBA treatment; PBA was well tolerated even over the 10-day treatment regimen. This observation is likely due to the rapid clearance of PBA, the ongoing replenishment of PHM by new protein synthesis, and the ability of peptide stores to sustain the functions of -amidated peptides during transient PHM inhibition. Our findings also indicate that intracellular PHM is somewhat protected from inhibition by PBA, especially in brain. Reductions on the order of 20 to 45% of control values were observed for hypothalamic PHM activity in animals whose circulating PHM activity was virtually eliminated by PBA treatment. Although PBA is highly lipophilic and can enter cells to inhibit PHM (Bradbury et al., 1990), its intracellular actions are likely to be impeded by both metabolism and the compartmentalization of PHM within secretory granules. In this regard, metabolism of PBA may explain the rebound of PHM activity in cultured CA77 cells despite the apparent continued presence of PBA as reported by Bradbury and coworkers (Bradbury et al., 1990). Under the conditions of their experiment, PBA may have been metabolized to an inactive metabolite that could not be distinguished from PBA itself. The differences in tissue sensitivity to PBA observed here may also be due to different catalytic states of PHM. Because inhibition of PHM by PBA requires active catalysis, tissues most affected by PBA treatment (e.g., atrium) may contain PHM in a more active catalytic state compared with tissues in which PHM is less affected by PBA administration (e.g., NIL). Finally, differential sensitivity to PBA could reflect varying levels of native peptide substrates that can protect PHM from inactivation by occupying its catalytic site (our unpublished findings).

The ability of PBA to inhibit PHM in vivo has important implications for the study of -amidation and the roles PHM may serve in addition to the bioactivation of peptide messengers. Specifically, recent evidence that PHM mediates the formation of primary fatty acid amides (Merkler et al., 1996), notably the sleep-inducing fatty acid amide oleamide (our unpublished findings), suggests that PHM may function in the bioactivation of both fatty acid and peptide messengers. Determining the physiological importance of these novel actions for PHM will be facilitated by the use of PBA as a pharmacological tool for predictably altering the functions of PHM in vivo.