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Biomedical subjects

G P Mueller

Publications and source records attributed to G P Mueller.

At least 19 recordsLinked to original sources
PubMed2000

Peptidylglycine-alpha-hydroxylating monooxygenase generates two hydroxylated products from its mechanism-based suicide substrate, 4-phenyl-3-butenoic acid.

The bifunctional enzyme peptidylglycine-alpha-amidating monooxygenase mediates the conversion of C-terminal glycine-extended peptides to their active alpha-amidated products. Peptidylglycine-alpha-hydroxylating monooxygenase (PHM, EC 1.14.17. 3) catalyzes the first reaction in this two-step process. The olefinic compound 4-phenyl-3-butenoic acid (PBA) is the most potent irreversible, mechanism-based PHM inactivator known. While the details of the inhibitory action of PBA on PHM remain undefined, covalent modification of the protein has been proposed as the underlying mechanism. We report here that, in the process of inactivating PHM, PBA itself serves as a substrate without covalently labeling the enzyme. Approximately 100 molecules of PBA are metabolized per molecule of PHM inactivated, under saturating conditions. The metabolism of PBA by PHM generates two hydroxylated products, 2-hydroxy-4-phenyl-3-butenoic acid and its allylic isomer, 4-hydroxy-4-phenyl-2-butenoic acid. While one enantiomer for each product is significantly favored in the reaction, both are produced. From these observations, we conclude that hydroxylated PBA products are formed by a delocalized free radical mechanism and that the lack of absolute stereospecificity indicates significant freedom of movement within the catalytic site. The ability of PHM to metabolize PBA suggests that the physiological functions of PHM may include the hydroxylation of substrates other than those containing terminal glycines.

Catalytic Domain↗
PubMed2000

Induction of peptidylglycine alpha-amidating monooxygenase in N(18)TG(2) cells: a model for studying oleamide biosynthesis.

The fatty-acid primary amide, oleamide, is a novel signaling molecule whose mechanism of biosynthesis is unknown. Recently, the N(18)TG(2) cell line was shown to synthesize oleamide from oleic acid, thereby demonstrating that these cells contain the necessary catalytic activities for generating the fatty-acid primary amide. The ability of peptide alpha-amidating enzyme, peptidylglycine-alpha-amidating monooxygenase (PAM; EC 1.14.17.3), to catalyze the formation of oleamide from oleoylglycine in vitro suggests this as a function for the enzyme in vivo. This investigation shows that N(18)TG(2) cells, in fact, express PAM and that cellular differentiation dramatically increases this expression. PAM expression was confirmed by the detection of PAM mRNA, PAM protein, and enzymatic activity that exhibits the functional characteristics of PAM isolated from mammalian neuroendocrine tissues. The regulated expression of PAM in N(18)TG(2) cells is consistent with the proposed role of PAM in the biosynthesis of fatty-acid primary amides and further establishes this cell line as a model for studying the pathway.

Animals↗
PubMed1999

Lactic acid does not directly activate hypothalamic-pituitary corticotroph function.

The role that metabolic products play in regulating the hypothalamic-pituitary-adrenal (HPA) axis during strenuous exercise is speculative. This investigation examined the extent to which lactic acid, a major metabolite of anaerobic exercise, directly affects hypothalamic-pituitary function. Specifically, beta-endorphin secretion was measured from AtT-20 (D-16) mouse corticotroph tumor cells treated either acutely (15 min - 180 min) or chronically (1 day - 3 day) with physiologic levels of lactate (0. 5 x 10-3 M to 5 x 10-2 M) or lactate in combination with the corticotroph releasing factors: corticotroph releasing hormone (CRH), arginine vasopressin (AVP), norepinephrine and/or epinephrine. Findings with AtT-20 cell cultures were shown to be representative of responses in primary cultures of rat anterior pituitary. Lactic acid did not alter the spontaneous release of beta-endorphin by AtT-20 cells under either acute or chronic conditions. While CRH, norepinephrine, and epinephrine evoked significant increases in beta-endorphin release, lactate, in combination with these secretagogues did not alter their effects. Similarly, lactic acid failed to alter basal or stimulated release of beta-endorphin by primary cultures of rat anterior pituitary. The addition of lactate (3 x 103 M) to rat hypothalamic explants did, however, produce a modest but significant reduction in spontaneous CRH release, suggesting that lactate may facilitate the return to basal secretion following exercise. The present findings show that physiologic concentrations of lactate have no effect, either alone or in combination with other pituitary secretagogues, on corticotroph secretion. Whereas a physiologic action for lactate within the hypothalamus is possible, the present findings indicate that lactate is an inhibitor of CRH release. Thus, lactate does not appear to play a direct role in the profound activation of the HPA axis that occurs in response to strenuous exercise.

Animals↗
PubMed1999

Differential regulation of peptide alpha-amidation by dexamethasone and disulfiram.

alpha-Amidation is essential for the function of many peptides in intercellular communication. This C-terminal modification is mediated in a two-step process by the hydroxylase and lyase activities of the bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The first step, catalyzed by peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17. 3), is rate limiting in the process, and therefore subject to regulation. Dexamethasone and disulfiram (tetraethylthiuram disulfide; Antabuse) were used as in vivo treatments to study the regulation of PHM expression and activity in cardiac atrium. Our findings show that both dexamethasone and disulfiram treatment increase the activity of PHM in atrial tissue but that they do so by distinctly different mechanisms. Dexamethasone elevated tissue levels of PAM mRNA and protein concurrently, suggesting that glucocorticoids regulate PAM expression at the level of gene transcription. In contrast, disulfiram treatment, which depletes stores of alpha-amidated peptides, increased the specific activity of PHM without affecting the level of PAM expression. The catalytic efficiency of PHM was enhanced by raising the Vmax of the enzyme. Importantly, this increase in Vmax was retained through purification to homogeneity, indicating that either a covalent modification or a stable conformational change had occurred in the protein. These novel findings demonstrate that the rate-limiting enzyme in the bioactivation of peptide messengers is differentially regulated by transcriptional and post-transcriptional mechanisms in vivo. It is proposed that regulation of PHM's expression and catalytic efficiency serve as coordinated physiologic mechanisms for maintaining appropriate levels of alpha-amidating activity under changing conditions in vivo.

Animals↗
PubMed1999

Captopril inhibits peptidylglycine- alpha-hydroxylating monooxygenase: implications for therapeutic effects.

The therapeutic actions of captopril are facilitated by its sulfhydryl moiety which interacts with the metal (Zn2+) prosthetic groups of angiotensin-converting enzyme (ACE; EC 3.4.15.1). This study focused on captopril as an inhibitor of another metal-dependent (Cu2+) enzyme, peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3). PHM is rate limiting in alpha-amidation, a COOH-terminal modification that bioactivates several pressor peptides. Captopril inhibited PHM in vitro in a dose-dependent manner with an IC50 of approximately 100 micromol/l. This inhibition was partially reversed by increased concentrations of Cu2+. Structurally similar nonsulfhydryl ACE inhibitors did not affect the activity of PHM. The present findings indicate that the therapeutic effectiveness of captopril may result from actions on a range of metalloenzymes including ACE and PHM.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

In vivo inhibition of peptidylglycine-alpha-hydroxylating monooxygenase by 4-phenyl-3-butenoic acid.

Peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17. 3) catalyzes the first and rate-limiting reaction in the two-step process that alpha-amidates neural and endocrine peptides. The substrate analog 4-phenyl-3-butenoic acid (PBA) was shown in vitro to selectively inhibit PHM without affecting the activity of peptidyl-alpha-hydroxyglycine alpha-amidating lyase, the enzyme that mediates the second reaction in alpha-amidation. Inhibition of PHM activity by PBA lowered the V(max) of the enzyme without altering its K(m). Administration of PBA in vivo profoundly inhibited serum PHM activity in a dose- and time-related fashion. Maximal reductions to less than 5% of control levels were observed 3 h after a single administration (500 mg/kg). Inhibition of serum PHM activity by PBA was short-lived, being fully reversed by 24 h postinjection. PHM activity in cardiac atrium, hypothalamus, and anterior and neurointermediate lobes of the pituitary were also decreased by PBA treatment but to a lesser extent than with serum. Inhibition of PHM activity by PBA was not cumulative over time when assessed 24 h after the last of 10 daily injections (500 mg/kg). The role of protein synthesis in maintaining PHM activity in blood was demonstrated by treatment with cycloheximide, which reduced serum PHM activity and retarded the recovery of PHM activity after PBA administration. It is concluded that the metabolism and/or clearance of PBA is rapid and that de novo protein synthesis has an important role in mediating the rapid restoration of PHM activity after PBA administration.

Animals↗
PubMed1997

Localization of putative calcium-responsive regions in the rat BDNF gene.

Brain-derived neurotrophic factor (BDNF) has potent trophic and protective actions on CNS neurons, including mesencephalic dopaminergic neurons, ventral forebrain cholinergic neurons and spinal motor neurons. To evaluate the effects of calcium and other second messengers on BDNF gene transcription, C6 glioma cells were treated for 4 h with the calcium ionophore A23187, forskolin + isobutyl-methyl-xanthine (IBMX), or the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate. Semi-quantitative RT-PCR analysis revealed that A23187 treatment increased BDNF transcripts containing the protein coding exon by 4.4-6.4-fold. Alternate BDNF transcripts were elevated to varying degrees after treatment with this ionophore and a subset of these transcripts was elevated following forskolin + IBMX treatment. When co-incubated with the RNA polymerase inhibitor, actinomycin D, A23187-induced increases were reduced or abolished, suggesting that calcium-mediated regulation of BDNF mRNA expression occurs at transcriptional as well as post-transcriptional levels. Transient transfection experiments employing reporter constructs containing serial 5' deletions of alternate BDNF promoters suggested that A23187-induced elevations in BDNF exon 1b, 1d and 1e containing transcripts are mediated by putative calcium-responsive regions flanking all three of these exons.

Animals↗
PubMed1997

Disulfiram causes sustained behavioral and biochemical effects in rats.

The present experiment examined effects of disulfiram (Antabuse) administration on behavioral measures of nociception (hot plate and tail flick), peripheral muscular performance (grip strength), motivated performance, balance, and coordination (rotorod) in 24 male Sprague-Dawley rats during and 2 wk after an eight-day administration of disulfiram. In addition, peptidylglycine 5(-hydroxylating monooxygenase (PHM) activity in several tissues and levels of alpha-amidated alpha-melanocyte stimulating hormone (alpha-MSH) in the neurointermediate lobe of the pituitary were assayed to evaluate biochemical effects of disulfiram. These particular assays were included because it has been reported that disulfiram affects alpha-amidated peptides via alteration of PHM activity. Decrements in all behavioral measures, except tail flick, occurred after one week of disulfiram administration. Decrements in grip strength continued for the 2 wk after cessation of disulfiram. Dose-related reductions in changes in PHM activity and levels of alpha-MSH were found 2 wk after cessation of disulfiram administration. The time course of the results suggest that changes in PHM activity may underlie decrements in grip strength. The present experiment provides a paradigm for further investigations of effects of alpha-amidated peptides on behavior.

Alcohol Deterrents↗
PubMed1997

Marked differences in functioning of the hypothalamic-pituitary-adrenal axis between groups of men.

To compare profiles of hypothalamic-pituitary-adrenal (HPA) responsiveness, healthy, moderately trained men (n = 15) were classified as high (n = 7) or low responders (n = 8) on the basis of plasma adrenocorticotropic hormone (ACTH) responses to strenuous treadmill exercise 4 h after 4 mg of dexamethasone (Dex). These groups were then evaluated to compare 1) HPA and growth hormone responses to exercise at 90% maximal oxygen uptake 4 h after placebo, Dex (4 mg), and hydrocortisone (100 mg); 2) pituitary-adrenal responses to infusion of arginine vasopressin (AVP); 3) plasma cortisol after a Dex suppression test (1 mg); and 4) behavioral characteristics. In comparison to low responders, high responders exhibited significantly 1) higher plasma ACTH responses to exercise after placebo and Dex; 2) higher plasma AVP secretion with exercise after placebo and marked Dex- and hydrocortisone-induced enhancement of exercise-induced AVP secretion; 3) lower Dex-induced increases in basal and stimulated growth hormone secretion; 4) higher plasma ACTH responses to infusion of AVP; and 5) a trend (P = 0.09) for higher trait anxiety ratings. Similar suppression of plasma cortisol was noted after 1 mg Dex. We conclude that subgroups of healthy male volunteers exhibit unique profiles of HPA responsiveness. We also believe that glucocorticoid pretreatment combined with strenuous exercise allows functional HPA responsiveness to be distinguished between subgroups of healthy controls and may be useful in the determination of susceptibility to disorders characterized by hyper- and hypo-HPA activation.

Adrenocorticotropic Hormone↗
PubMed1995

Peptide alpha-amidation: differential regulation by disulfiram and its metabolite, diethyldithiocarbamate.

The rate limiting step in the alpha-amidation of bioactive peptides is catalyzed by peptidylglycine-alpha-hydroxylating mono-oxygenase (PHM; EC 1.14.17.3). Sustained treatment with disulfiram (Antabuse), the disulfide dimer of diethyldithiocarbamate (DDC), inhibits PHM in vivo, causing tissue levels of alpha-amidated peptides to decrease. As a compensatory response, PHM protein is modified in such a way that its activity is increased when assayed under optimal conditions in a test tube. Because disulfiram is rapidly reduced to DDC in vivo, this investigation sought to determine if metabolic transformation plays a role in the effects of disulfiram treatment on alpha-amidation. While disulfiram treatment reduced concentrations of alpha-amidated peptides in the pituitary neurointermediate lobe and brain, DDC treatment did not, even at comparatively high doses. Both treatments increased the activity of PHM extracted from the neurointermediate pituitary and assayed under optimal copper conditions in vitro. Only disulfiram treatment elicited an increase in PHM activity extracted from cardiac atrium. It is concluded that the activity of PHM is regulated in a tissue specific fashion and that not all of the actions of disulfiram require its metabolism to DDC.

Animals↗
PubMed1995

Surgical infections in AIDS patients.

Many patients with acquired immune deficiency syndrome (AIDS) and abdominal pain are evaluated by the surgeon, and the majority have gastroenteritis, which can be treated with specific antimicrobials. There are some, however, who need more extensive investigation or who have an intra-abdominal infective process that requires surgical treatment. The one and a half decades of experience with human immunodeficiency virus (HIV) and AIDS has defined the role of the surgeon in treating patients with HIV. Major infective processes that may require surgical involvement include cytomegalovirus infection of the intestinal tract; appendicitis, which may be due to opportunistic infections; spontaneous bacterial peritonitis; cholecystitis; and obstructive jaundice with underlying sclerosis of the biliary tree. Early diagnosis and prompt surgical treatment are critical in the management of HIV-infected patients. For example, cytomegalovirus affecting the gastrointestinal tract may lead to perforation with the development of generalized fecal peritonitis; the clinical presentation of acute appendicitis in HIV patients may not include the usual rise in white blood cell count; and bacterial peritonitis in patients with AIDS may be caused by opportunistic pathogens or, as in the classical case, a single gram-negative bacillus or pneumococcus. This review article focuses on intra-abdominal infections in patients with HIV and AIDS.

Abdominal Pain↗
PubMed1994

Alternate 5' exons in the rat brain-derived neurotrophic factor gene: differential patterns of expression across brain regions.

Brain-derived neurotrophic factor (BDNF) enhances the survival of dopaminergic neurons and protects them from neurotoxins in vitro. This trophic factor might thus be of therapeutic value for the treatment of Parkinsonian syndromes. The rat BDNF gene consists of several upstream noncoding exons that are alternatively spliced to a common coding exon. To investigate BDNF 5' exons expressed in the adult rat brain, we subjected RNA from cerebellum to 5'-RACE analysis and compared the resulting clones to previously reported 5' exon sequences from rat brain and hippocampus. In addition to known 5' exons, we isolated a BDNF transcript with a novel 5' sequence representing yet another alternate upstream exon in this gene. Quantitative PCR analysis of BDNF mRNAs containing each of the five upstream exons indicated that each of the alternate transcripts is most abundant in the hippocampus, intermediate in the substantia nigra and cerebellum and least abundant in the striatum. However, the magnitude of these differences in expression varied considerably suggesting that BDNF gene transcription in the mature brain is regulated by alternate promoters that are differentially active across regions.

Alternative Splicing↗
PubMed1994

Regulated endocrine-specific protein-18: a short-lived novel glucocorticoid-regulated endocrine protein.

Regulated endocrine-specific protein-18 (RESP18) is an 18-kilodalton endocrine-specific transcript whose expression is regulated by a number of different physiological and pharmacological stimuli in different tissues. RESP18 messenger RNA was identified in all cell types in the anterior pituitary, at levels that varied 2-fold from the lowest (corticotropes and thyrotropes) to the highest (gonadotropes, somatotropes, and mammotropes); the melanotropes of the intermediate pituitary have levels of RESP18 messenger RNA comparable to the highest levels in cells in the anterior pituitary. Mouse RESP18 was cloned and used as the basis for biosynthetic studies on RESP18 in AtT-20 cells, which express RESP18 endogenously; mouse RESP18 was highly homologous to rat RESP18. Pulse-chase biosynthetic labeling studies showed that AtT-20 cells expressed much less RESP18 than the endogenous prohormone, POMC, but that glucocorticoid treatment lowered POMC and raised RESP18 biosynthetic rates so that they were nearly equimolar. Surprisingly, RESP18 was not processed to smaller peptides to any significant extent, nor was RESP18 or any smaller peptide secreted. Newly synthesized RESP18 normally disappeared from AtT-20 cell extracts with a half-life of less than 15 min; the intracellular half-life of RESP18 was increased strikingly after glucocorticoid treatment of the cells. Upon subcellular fractionation, RESP18 was found to be entirely particulate and to cofractionate with markers for the endoplasmic reticulum, rather than with markers for secretory granules, such as POMC and prohormone-processing enzymes. Therefore, RESP18 is a major glucocorticoid-responsive protein in the secretory pathway of corticotropes, but its function may be entirely within the neuroendocrine cell.

Amino Acid Sequence↗
PubMed1994

Exercise-induced activation of the hypothalamic-pituitary-adrenal axis: marked differences in the sensitivity to glucocorticoid suppression.

Treadmill exercise activates the hypothalamic-pituitary-adrenal axis and evokes metabolic responses proportional to exercise intensity and duration. To determine whether glucocorticoid administration would alter humoral and metabolic regulation during exercise, we administered 4 mg dexamethasone (DEX) or placebo to 11 normal, moderately trained men (19-42 yr old) in a double blinded random fashion 4 h before high intensity intermittent treadmill running. Plasma levels of ACTH, cortisol, arginine vasopressin (AVP), lactate, and glucose were measured before, during, and after exercise. A wide range of ACTH responses were seen in the DEX-treated group and arbitrarily defined as two subsets of individuals according to their responses to dexamethasone: DEX nonsuppressors and DEX suppressors. Exercise-induced increases in heart rate and circulating concentrations of cortisol, AVP, lactate, and glucose were all significantly greater (P < 0.05) in nonsuppressors (n = 4) compared to suppressors (n = 7) after both placebo and DEX administration. Interestingly, heart rate, AVP, and lactate responses were unaltered by DEX alone in both groups. In summary, this study demonstrates that normal individuals exhibit differential neuroendocrine and metabolic responses to exercise and pituitary/adrenal suppression after pretreatment with DEX. These findings reflect marked individual differences in the stress response to exercise that may derive from or lead to differential glucocorticoid negative feedback sensitivity in humans.

Adrenal Glands↗
PubMed1993

Induction of peptidylglycine alpha-hydroxylating monooxygenase activity by nerve growth factor in PC12 cells.

Pheochromocytoma PC12 cells grown in the presence of nerve growth factor (NGF) undergo marked neuronal differentiation. During this process gene expression is altered, resulting in the activation of genes specific for neuronal properties, including the gene encoding neuropeptide Y (NPY). Here we sought to determine whether NGF also induces the activity of peptidylglycine alpha-hydroxylating monooxygenase (PHM) (EC1.4.17.3). PHM catalyzes the rate limiting step in the formation of alpha-amidated NPY from its glycine extended precursor, a posttranslational modification essential for biologic activity. PC12 cells were grown with or without NGF and assayed for PHM activity under optimal conditions. Whole cell extracts, medium and soluble and membrane bound fractions were assayed; total cellular PHM activity was found to be primarily membrane bound (fivefold greater than in soluble) and very little activity was released into the medium. Compared to control cells, PHM activity was increased significantly by NGF by 24 h but not before 4 h exposure. Through kinetic analysis, it was determined that the NGF-induction of PHM was a result of an increase in Vmax with no change in Km. It was found that the glucocorticoid, dexamethasone (DEX), decreased basal PHM activity and prevented its induction by NGF. Cotreatment with DEX for up to 7 d, however, did not dramatically alter the pronounced changes in cell morphology that occurred in response to NGF. These findings indicate that NGF and glucocorticoids exert reciprocal control over the activity of PHM in PC12 cells. As such, the process of differentiation in PC12 cells is a model for studying the mechanisms that coordinate the expression and activity of peptide processing enzymes with the regulation of their substrates and products.

Amino Acid Sequence↗
PubMed1993

Peptide alpha-amidation and peptidylglycine alpha-hydroxylating monooxygenase: control by disulfiram.

The final two steps in the biosynthesis of alpha-amidated bioactive peptides are catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5). These enzymes are derived from the bifunctional precursor protein, peptidylglycine alpha-amidating monooxygenase. Because PHM is rate-limiting in peptide amidation and is copper-dependent, we examined the consequences of in vivo treatments with the copper-chelating drug disulfiram (Antabuse) on levels of alpha-amidated peptides and expression of PHM and PAL. Decreases in two amidated peptides (alpha-melanotropin and cholecystokinin) after disulfiram treatment were extremely pronounced outside the blood-brain barrier, with moderate decreases in the central nervous system. Unexpectedly, when assayed under optimal conditions in vitro, PHM activity was increased by disulfiram treatment, whereas PAL activity was unaltered. The increase in PHM activity in pituitary and atrium occurred within a few hours after the start of disulfiram treatment and was sustained up to 2 weeks after the cessation of treatment, whereas levels of alpha-amidated peptides remained low. Northern and Western blot analyses demonstrated that disulfiram had no influence on levels of peptidylglycine alpha-amidating monooxygenase mRNA or protein. Thus, inhibition of alpha-amidation by disulfiram in vivo occurs despite an increased Vmax of PHM assayed in vitro. The increase in PHM activity may result from induction of a physiologic mechanism that normally regulates this rate-limiting enzyme.

Amides↗
PubMed1992

N-acetylation and C-terminal proteolysis of beta-endorphin in the anterior lobe of the horse pituitary.

beta-Endorphin is post-translationally processed to both N-acetylated and C-terminally shortened derivatives in the anterior lobe of the horse pituitary, a processing pattern qualitatively different from that of the rat and virtually every other mammalian species. Thus, separation of the molecular forms of beta-endorphin using gel filtration and ion exchange chromatography showed that the horse anterior lobe primarily contains beta-endorphin-1-31 and N-acetyl-beta-endorphin-1-27 along with smaller amounts of beta-lipotropin, beta-endorphin-1-27, and N-acetyl-beta-endorphin-1-31 and -1-26, in contrast to the rat anterior lobe, which contains approximately equal amounts of beta-lipotropin and beta-endorphin-1-31. Immunohistochemical experiments using an antiserum which specifically recognizes N-acetylated beta-endorphin peptides confirmed that N-acetyl-beta-endorphin immunoreactivity is present in the anterior lobe of the horse, but not the rat. The intermediate lobe of both species primarily synthesizes N-acetylated, C-terminally shortened beta-endorphin peptides, and while distinct species differences do occur, they were relatively minor, consisting of quantitative differences in the relative proportion of each peptide. These results are consistent with earlier reports that beta-endorphin processing in the rat pituitary is tissue specific; the anterior and intermediate lobes produce entirely different sets of beta-endorphin peptides. In the equine pituitary, however, both pituitary lobes produce the same multiple beta-endorphin forms, possessing both opioid and nonopioid properties, although their relative amounts differ.

Acetylation↗