Chemical modification of dopamine beta-hydroxylase

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.     

Soluble and membrane-bound forms of dopamine beta-hydroxylase are encoded by the same mRNA.

A full length cDNA clone for bovine dopamine beta-hydroxylase was expressed in rat pheochromocytoma PC12 cells by stable transformation of this cell line with a plasmid expression vector. The recombinant protein exhibited dopamine beta-hydroxylase enzyme activity and was found in both the soluble and membrane fractions of the secretory vesicle. Immunoprecipitation of cell extracts from recombinant cell lines with dopamine beta-hydroxylase antisera followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two subunits, which migrated to relative molecular masses of 76 and 78 kDa. The recombinant protein co-fractionated with neurotransmitter when subcellular structures were separated by sucrose gradient density centrifugation, suggesting that the protein was routed to the secretory vesicles. Dopamine beta-hydroxylase immunoreactivity in those sucrose gradient fractions presumed to contain secretory vesicles was resistant to treatment with trypsin unless the nonionic detergent Triton X-100 was also present to disrupt membrane structure. The 76- and 78-kDa isoform were each found in both the membrane and soluble fractions of the secretory vesicle. Treatment of cultured cells with nerve growth factor or 8-(4-chlorophenylthio)-cyclic AMP alters the relative distribution of the subunits such that the 76-kDa form predominates. The subcellular distribution of a dopamine beta-hydroxylase cDNA clone lacking the first 16 nucleotide residues was also determined. The predicted amino acid sequence of the protein encoded by this cDNA would be deleted of the first 13 residues of the signal sequence, which were reported to be present in the membrane-bound form, but not the soluble form, of native dopamine beta-hydroxylase (Taljanidisz, J., Stewart, L., Smith, A. J., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061). Immunoprecipitable dopamine beta-hydroxylase derived from expression of the deleted cDNA was found in both the membrane-bound and soluble fractions of the secretory vesicle. These experiments demonstrate that the membrane-bound and soluble forms of dopamine beta-hydroxylase are derived from one primary translation product, which is also sufficient to produce enzyme activity. In addition, the amino-terminal amino acids encoding residues 1-13, which compose the hydrophilic region of the signal sequence, are not necessary for the biogenesis of membrane-bound dopamine beta-hydroxylase.     

The membrane-binding segment of dopamine beta-hydroxylase is not an uncleaved signal sequence

Dopamine beta-hydroxylase exists in bovine adrenal medulla chromaffin granules in both soluble and membrane-bound forms. The mechanism by which membranous dopamine beta-hydroxylase is bound to granule membranes has been elusive. Recently, evidence that covalently attached phosphatidylinositol does not serve as an anchor for membranous dopamine beta-hydroxylase was reported (Stewart, L. C., and Klinman, J. P. (1988) J. Biol. Chem. 263, 12183-12186). It was suggested that an uncleaved signal sequence could serve as a mode of attachment for the membrane-bound hydroxylase. Amino-terminal sequence analysis of purified bovine membranous dopamine beta-hydroxylase demonstrates that this form of the enzyme possesses an amino-terminal sequence similar to the soluble enzyme. Additionally, the 75- and 72-kDa bands of membranous dopamine beta-hydroxylase were electrophoretically eluted from a preparative sodium dodecyl sulfate-polyacrylamide gel and sequenced. Both bands had the amino-terminal sequence characteristic of the soluble bovine enzyme. These sequence results eliminate the possibility that an uncleaved signal sequence serves as the membrane anchor.     

Properties of partially purified bovine brain dopamine beta-hydroxylase.

Dopamine beta-hydroxylase has been partially purified from bovine brain. A 140-fold purification factor was achieved using solubilization with Triton X-100, ammonium sulphate fractionation between 20-50 per cent saturation, affinity chromatography on concanavalin A-Sepharose 4 B and then filtration through Sephadex G200. The specific activity at the end was 51 nmoles/h/mg protein. The majority of endogenous inhibitors were lost. Immunological studies, kinetic studies, studies on the interaction with lectins and the effect of carboxylic acids on enzyme activity were carried out. Our data are in favour of the close similarity between the bovine brain and adrenal enzymes. No major differences could be found, at least with the characterization experiments using in the present study.     

Blood dopamine beta-hydroxylase activity and noradrenaline levels in the developing white rat.

Dopamine beta-hydroxylase (DBH) (3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating) (EC 1.14.17.1) activity in serum of blood obtained by decapitation of white rats at 19, 20, and 21 days in utero, immediately after birth, and postnatally to 70 days, was measured. Noradrenaline (NA) and DBH in plasma from undisturbed, cannulated, postweaning rats were also assayed. During the last few days in utero and the first 2 postnatal days serum DBH activity tripled and then remained elevated during the suckling period. Upon weaning, serum DBH activity declined at first precipitously and then more slowly, until the adult level was reached around 70 days of age. This postweaning decrease in DBH activity was also observed with the cannulated animals. In contrast, plasma NA levels remained low and constant throughout the postweaning period. In suckling rats treated with 6-hydroxydopamine from 2 to 12 days of age, serum DBH activity decreased to less than half its initial value by day 8. It is suggested that the observed changes in serum DBH activity in fetal and postnatal rats reflect ontogenetic changes in sympathetic nerve terminals and that they are probably not correlated with release of NA.     

Complexes of chromogranin A and dopamine beta-hydroxylase among the chromogranins of the bovine adrenal medulla

1. The core proteins of chromaffin granules have been examined by polyacrylamide gel electrophoresis and crossed immunoelectrophoresis against monospecific antisera. 2. Dopamine beta-hydroxylase (dopamine beta-monooxygenase, EC 1.14.17.1) appeared as the major immunogen of the core proteins and accounted for 4 and 8% by weight of the crude lysate and membrane-containing fractions, respectively. 3. The non-ionic detergent, Berol, solubilized dopamine beta-hydroxylase from the membranes in a form which was immunologically identical but of lower relative mobility by crossed immunoelectrophoresis. In the absence of detergent a difference in relative mobility was also noted between the purified enzyme and that contaminated by chromogranin A. These observations suggest that several molecular forms of dopamine beta-hydroxylase may occur which differ in size and/or charge due to interactions with the contaminants under the experimental conditions. 4. The main chromogranin in the crude lysate was absent from electropherograms of the acidic chromogranins (95--96% of total protein in lysate). These were obtained free of dopamine beta-hydroxylase by concanavalin A adsorption at high ionic strength or by acidification in 2 M acetic acid. The main band reappeared upon recombination with dopamine beta-hydroxylase, indicating the presence of some dopamine beta-hydroxylase, possibly as dimers, in this main, chromogranin A band. A protein concentration-dependent aggregate of dopamine beta-hydroxylase-free chromogranin A was detected, with a relative mobility slightly faster than the main band of the crude lysate.     

Role of ascorbic acid in dopamine beta-hydroxylation. The endogenous enzyme cofactor and putative electron donor for cofactor regeneration

The role(s) of ascorbic acid in dopamine beta-hydroxylation was studied in primary cultures of bovine adrenomedullary chromaffin cells and in isolated bovine adrenomedullary chromaffin vesicles. Dopamine beta-hydroxylase activity was assessed by measuring the rate of conversion of tyramine to octopamine. The ascorbic acid content of chromaffin cells declined with time in culture and the dopamine beta-hydroxylase activity of ascorbate-depleted cells was low. Ascorbate additions to ascorbate-depleted cells increased both the intracellular ascorbate concentrations and the rates of dopamine beta-hydroxylation. Ascorbate uptake into the cells was rapid; however, the onset of enhanced octopamine synthesis by added ascorbate was delayed by several hours and closely followed the time course for accumulation of the newly taken up ascorbate into the chromaffin vesicle. The amount of octopamine synthesized by the chromaffin cells exceeded the intracellular ascorbate content and ascorbate levels were maintained during dopamine beta-hydroxylation in the absence of external ascorbate. This suggests an efficient recycling of ascorbate. In contrast to intact cells, ascorbic acid was depleted during octopamine synthesis in isolated chromaffin vesicles. The molar ratio of octopamine formed to ascorbate depleted was close to unity. Thus, the recycling of intravesicular ascorbate depends on an extravesicular factor(s). The depletion of intravesicular ascorbate during dopamine beta-hydroxylation was prevented by the addition of nonpermeant extravesicular electron donors such as ascorbate or glucoascorbate. This suggests that intravesicular ascorbate is maintained in the reduced state by electron transport across the vesicle membrane. These results are compatible with the hypothesis that both intra- and extravesicular ascorbate participate in the regulation of dopamine beta-hydroxylase. Intravesicular ascorbate is the cofactor for the enzyme. Cytosolic ascorbate is most likely the electron donor for the vesicle-membrane electron transport system which maintains the intravesicular cofactor concentration.     

Isolation and reconstitution of the membrane-bound form of dopamine beta-hydroxylase

Dopamine beta-hydroxylase is present in the bovine adrenal medulla in two forms, soluble and membrane bound. Previous isolation procedures for the membranous hydroxylase have resulted in a form of enzyme identical in subunit structure with the soluble type. We report here the isolation of a membrane-bound form of dopamine beta-hydroxylase which is structurally different from the soluble form. The isolated membranous enzyme has a large apparent molecular weight on gel filtration, is amphiphilic, and contains bound phospholipid which is predominantly phosphatidylserine. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows that the membranous hydroxylase contains two nonidentical subunits under both reducing and nonreducing conditions. Under reducing conditions the apparent molecular weights of the two subunits are 70,000 and 75,000 and both contain carbohydrate. The purified membranous hydroxylase binds to phospholipid vesicles and chymotryptic digestion of the bound enzyme suggests that two forms of the membranous hydroxylase exist.     

Disorders of the aldosterone synthase and steroid 11beta-hydroxylase deficiencies

The most potent corticosteroids are 11beta-hydroxylated compounds. In humans, two cytochrome P450 isoenzymes with 11beta-hydroxylase activity, catalysing the biosynthesis of cortisol and aldosterone, are present in the adrenal cortex. CYP11B1, the gene encoding 11beta-hydroxylase (P450c11), is expressed on high levels in the zona fasciculata and is regulated by ACTH. CYP11B2, the gene encoding aldosterone synthase (P450c11Aldo), is expressed in the zona glomerulosa under primary control of the renin-angiotensin system. Aldosterone synthase has 11beta-hydroxylase activity as well as 18-hydroxylase activity and 18-oxidase activity. The substrate for CYP11B2 is 11-deoxycorticosterone, that of CYP11B1 is 11-deoxycortisol. Mutations in CYP11B1 cause congenital adrenal hyperplasia (CAH) due to 11beta-hydroxylase deficiency. This disorder is characterized by androgen excess and hypertension. Mutations in CYP11B2 cause congenital hypoaldosteronism (aldosterone synthase deficiency) which is characterized by life-threatening salt loss, failure to thrive, hyponatraemia and hyperkalaemia in early infancy. Both disorders have an autosomal recessive inheritance. Classical and nonclassical forms of 11beta-hydroxylase deficiency can be distinguished. Studies in heterozygotes for classical 11beta-hydroxylase deficiency show inconsistent results with no or only mild hormonal abnormalities (elevated plasma levels of 11-deoxycortisol after ACTH stimulation). In infants with congenital hypoaldosteronism, a comparable frequency of 18-hydroxylase deficiency (aldosterone synthase deficiency type I) and of 18-oxidase deficiency (aldosterone synthase deficiency type II) can be found. Molecular genetic studies of the CYP11B1 and CYP11B2 genes in 11beta-hydroxylase deficiency or aldosterone synthase deficiency have led to the identification of several mutations. Transfection experiments showed loss of enzyme activity in vitro. In some of the patients with 18-oxidase deficiency (aldosterone synthase deficiency type II) no mutations in the CYP11B2 gene were identified. Refined methods for steroid determination are the basis for the diagnosis of inborn errors of steroidogenesis. Molecular genetic studies are complementary; on the one hand, they have practical importance for the prenatal diagnosis of virilizing CAH forms and on the other hand, they are of theoretical importance in terms of our understanding of the functioning of cytochrome P450 enzymes. Copyrightz1999S.KargerAG, Basel     

Mechanism of biosynthesis of soluble and membrane-bound forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells

Dopamine beta-hydroxylase was present as 2 subunit forms (apparent Mr = 77,000 and 73,000) in the PC12 pheochromocytoma cell line as detected by immunoprecipitation from [35S]methionine-labeled cultures, and analyzed by sodium dodecyl sulfate gel electrophoresis and fluorography. The Mr = 77,000 form was present in a crude membrane fraction, while the Mr = 73,000 form was soluble. Both forms appeared to be present in approximately equal amounts, and both were glycosylated. Treatment of PC12 cells with tunicamycin, a potent inhibitor of core glycosylation in the endoplasmic reticulum, completely inhibited the appearance of the Mr = 77,000 and Mr = 73,000 forms, and 2 new immunoreactive polypeptides were obtained (apparent Mr = 67,000 and 63,000). Pulse-chase experiments suggested that the Mr = 77,000 form is initially synthesized (by 5 min) and a portion is converted in 15-90 min to the Mr = 73,000 form. Thereafter, the ratio between forms remains relatively constant, at least for several hours. Translation of mRNA from bovine and rat adrenals, and immunoprecipitation, indicated that dopamine beta-hydroxylase is initially synthesized as a single polypeptide (apparent Mr = 67,000). The subcellular site of biosynthesis of dopamine beta-hydroxylase was determined by isolation of mRNA from free and membrane-bound polysomes from bovine adrenal medulla. Translation in a cell free system and immunoprecipitation localized the synthesis of dopamine beta-hydroxylase on membrane-bound polysomes. These experiments suggest that both soluble and membrane-bound forms of dopamine beta-hydroxylase are synthesized and core glycosylated in the endoplasmic reticulum, and that there probably is a precursor-product relationship between the Mr = 77,000 and the Mr = 73,000 subunit forms of dopamine beta-hydroxylase.     

Multiple forms of human dopamine beta-hydroxylase in SH-SY5Y neuroblastoma cells

Dopamine beta-hydroxylase exists as three forms in human neuroblastoma (SH-SY5Y) cells. The membrane-bound form of the hydroxylase contains three different species with apparent relative molecular weights of 73,000, 77,000, and 82,000. The intracellular soluble form of dopamine beta-hydroxylase was present as a single species with an apparent molecular weight of 73,000. Pulse-chase experiments showed that membranous dopamine beta-hydroxylase contains two subunit forms of 73,000 and 77,000 after short chase times. The soluble hydroxylase was synthesized as a single species of 73,000 at approximately the same rate as the lower molecular weight species of the membranous enzyme. A constitutively secreted third form of the enzyme with an intermediate apparent molecular weight also incorporated [35S]sulfate, whereas no significant amount of [35S]sulfate was observed in the cellular forms of the enzyme. The [35S]sulfate was incorporated on N-linked oligosaccharides. Approximately 12% of the enzyme is released constitutively within 1 h. These results demonstrate that neuronal cells have the ability to constitutively secrete a specific form of dopamine beta-hydroxylase which may contribute to the levels of this enzyme found in plasma.     

Changes in adrenal dopamine concentration after metyrapone or ACTH administration: implications for the in vivo regulation of dopamine beta-hydroxylase by glucocorticoids.

$\text{In vitro}$ studies have demonstrated that rat adrenal dopamine beta-hydroxylase activity is controlled by neural input and by glucocorticoid production. However, beta-hydroxylation of dopamine $\text{in vivo}$ is a first-order reaction and may be considerably slower than the maximal rate determined by $\text{in vitro}$ methods. To estimate the $\text{in vivo}$ reaction rate the concentrations of dopamine (substrate) and of beta-hydroxylated catecholamine (product) were measured as a function of endogenous glucocorticoid production. Beta-hydroxylated catecholamine changed little but dopamine was increased 2-fold or more 17.5 h after the inhibition of steroidogenesis with metyrapone. Dopamine was also increased by metyrapone in animals with pre-existing adrenal denervation. ACTH 17.5 h before sacrifice caused only slight changes in normal rats but reduced the increase in dopamine caused by stress. The results indicate that adrenal dopamine concentration is inversely related to glucocorticoid production at a given level of neural input and provide $\text{in vivo}$ evidence that glucocorticoids maintain dopamine beta-hydroxylase activity in the adrenal gland.     

Hip fractures in elderly--ten years analysis

Geriatric hip fractures (GHP) are the major problem in the geriatric traumatology and it is estimated that treatment of GHF will spend a large amount of health care resources. The aim of this retrospective study was to compare differences in incidence of operatively treated patients with GHF, type of treatment depending of the type of fracture, early postoperative mortality, length of stay and costs of used implants within a 10 years period. Surgically treated 2478 patients, older than 65 years with hip fractures were included in the study. Patients were grouped according to the type of fracture (femoral neck fracture or intertrochanteric femoral fracture) and used implant. Results showed increasing trend in GHF in our County in the last 10 years. There was a shifting trend in used implants, and new surgical techniques were used more commonly in the last few years. In observed period there were no significant changes in revision surgery and length of hospital stay. The mortality decreased, especially in males, but generally it was not in correlation with used implant. At the 10-years period increase in patients with GHF of 179% was followed with 4 time higher increase in implant prices. Present reimbursement in health care system does not calculate the difference of implant costs in hospital expenses, therefore proper usage of modern implants and careful planning in the treatment of GHF is necessary.     

Immunoelectron microscopic localization of dopamine beta-hydroxylase and chromogranin A in adrenomedullary chromaffin cells

Dopamine beta-hydroxylase (DBH) was purified from bovine adrenal medullae. Rabbit IgG raised against DBH inhibited its activity by 80%. In an immunoblot analysis, the IgG specifically recognized two subunits of DBH the 72 and 75 KD components. Chromogranin A (CGA) also was purified from bovine adrenal medullae, and rabbit IgG against CGA recognized this chromogranin A in the immunoblot analysis. The intracellular distribution of DBH and CGA in bovine chromaffin cells was determined quantitatively by immunoelectron microscopy using post-embedding protein A-gold technique. DBH and CGA were localized exclusively on chromaffin granules. The binding of gold particles to these granules was saturable. The maximum number of gold particles bound to the granules roughly corresponded to the number of DBH or CGA molecules in the granules estimated biochemically. DBH was observed evenly in the periphery and in the dense matrix of the chromaffin granules.     

Human plasma dopamine beta-hydroxylase. Purification and properties

Dopamine beta-hydroxylase was isolated from normal human plasma. The major form of the active enxyme in plasma was purified to apparent homogeneity and is a 300,000-dalton tetramer containing 4 atoms of tightly bound copper. About 20% of the enzyme activity in plasma was isolated as a dimeric form of this enzyme. Sodium dodecyl sulfate gel electrophoresis of the purified form gave a polypeptide subunit molecular weight of 72,000 and disulfide-linked dimers of this component were observed. Both forms of the enzyme are apparently glycoproteins and interact with immobilized concanavalin A. Furthermore, the enzyme is capable of binding to alkyl-substituted agarose by hydrophobic interaction. Advantage was taken of these properties to purify the enzyme. Both purified tetramer and partially purified dimer were further characterized by kinetic analysis and the Stokes radii and S20,W of these species were compared. Rabbit antiserum to the purified tetramer revealed no immunochemical differences between the two enzyme forms by using a method of immunotitration.     

Effect of monensin on synthesis, post-translational processing, and secretion of dopamine beta-hydroxylase from PC12 pheochromocytoma cells

Monensin was used to ascertain the location in the biosynthetic pathway where the 77,000-Mr membrane-bound subunit form of dopamine beta-hydroxylase is post-translationally converted to the 73,000-Mr soluble form. Treatment with low concentrations of monensin (less than or equal to 50 nM) completely depleted the cells of the norepinephrine and dopamine, had a small effect on protein synthesis, and enhanced post-translational processing of only dopamine beta-hydroxylase which was previously synthesized and presumably packaged into neurosecretory vesicles. At these low concentrations, exit from the Golgi apparatus did not appear to be blocked since stimulated secretion of a group of high molecular weight [35S]methionine-labeled proteins was not inhibited. Treatment with higher concentrations of monensin (200 nM) prevented the secretion of the [35S] methionine-labeled proteins normally released with a secretagogue, and also prevented the secretion of [3H] mannose-labeled proteins including dopamine beta-hydroxylase. Surprisingly, a group of lower molecular weight [35S]methionine-labeled proteins was now released from monensin-treated cells. Treatment with high concentrations of monensin (greater than or equal to 200 nM) appeared to block the secretory pathway prior to the packaging step, probably in the Golgi apparatus. If the proteins were packaged prior to monensin treatment, they were released upon stimulation with secretagogues. Monensin treatment (200 nM) enabled the post-translational processing of newly synthesized dopamine beta-hydroxylase, from the 77,000-Mr to the 73,000-Mr subunit form, to go to completion. The susceptibility of this 73,000-Mr subunit form to endoglycosidase H digestion was unaltered, suggesting that dopamine beta-hydroxylase from monensin-treated cells may have the same high mannose oligosaccharide content as native dopamine beta-hydroxylase. These experiments indicate that the post-translational processing of dopamine beta-hydroxylase occurs in the Golgi apparatus and may continue in immature granules prior to their acidification.     

Orthognathic surgery in cleft patients treated by early bone grafting

For the past 25 years at Children's Memorial Hospital in Chicago a protocol has been followed for complete clefts that involves placement of an infant maxillary orthopedic appliance prior to lip closure, surgical closure of the lip, autogenous split-rib grafts to the alveolus to stabilize maxillary segments, and palatal closure, generally within the first year of life. The oldest 36 patients whose skeletal growth was for all practical purposes finished have been followed to determine the need for and type of orthognathic surgery. Of the total sample, 8 patients (22.2 percent) required some type of sagittal orthognathic surgery (1 patient in this group also required vertical maxillary alignment) and 2 patients required maxillary augmentation only in the form of an onlay graft. This report may serve as a baseline for others who wish to report on the incidence and type of orthognathic surgery in their cleft palate centers.     

Mechanisms by which dopamine alters blood flow distribution during lobar collapse in dogs

Dopamine increases blood flow to a hypoxic left lower lobe in dogs. To elucidate possible mechanisms, left lower lobe collapse was induced in anesthetized dogs, and lobar (QLLL) and total (QT) pulmonary blood flow was measured by electromagnetic flow probes. Dopamine infusion increased mean pulmonary arterial pressure (Ppa), QT, and QLLL. However, the increase in QLLL was double that produced by a similar increase in Ppa without increase in QT (inflation of a Swan-Ganz balloon in right pulmonary artery) or by a similar increase in QT with smaller increase in Ppa (opening of arteriovenous fistulas). QLLL/QT was not changed by opening arteriovenous fistulas, but was increased by Swan-Ganz balloon inflation, and by infusion of dopamine. It is concluded that the increase in QLLL/QT produced by dopamine was due to a decrease in hypoxic vasoconstriction in the lobe secondary to an increase in mixed venous PO2 and to vasoconstriction in the oxygenated lung.     

Evaluating postoperative analgesics in mice using telemetry

The study examined the efficacy of preemptive or postoperative analgesia on surgical pain in the mouse. Radiotelemetry transmitters were surgically implanted in 28 female ICR mice. A mock ova implantation surgery was then performed. Mice were treated with a single dose of buprenorphine or flunixin meglumine prior to or after surgery, three doses of buprenorphine, or were untreated. Heart rate, blood pressure, home cage activity, food and water consumption, and body weight were measured. The no-analgesia group showed no significant differences between any parameters collected prior to surgery and those collected at similar times during the day of surgery. Significant increases in mouse activity on the day of surgery occurred with all analgesic treatments, compared with pre-surgical activity. There were no consistent significant changes in any other telemetry parameter after treatment with analgesics compared with no analgesia. Food consumption and body weight the day after surgery were reduced significantly in the animals treated with three doses of buprenorphine compared with untreated mice and mice given a single dose of buprenorphine. We conclude that the mock ova implant procedure does not induce sufficient pain to cause alterations in heart rate and blood pressure in the mouse. Activity was significantly reduced in the first 6 h after surgery in mice without analgesia, compared with activity prior to surgery. There were no significant differences between pre-emptive and postoperative analgesia. Body weight and food and water consumption were poor measures of pain because analgesia alone affected these parameters.