PRECISION: the Belgian molecular profiling program of metastatic cancer for clinical decision and treatment assignment.

PRECISION is an initiative from the Belgian Society of Medical Oncology (BSMO) in collaboration with several stakeholders, encompassing four programs that aim to boost genomic and clinical knowledge with the ultimate goal to offer patients with metastatic solid tumors molecularly guided treatments. The PRECISION 1 study has led to the creation of a clinico-genomic database. The Belgian Approach for Local Laboratory Extensive Tumor Testing (BALLETT) and GeNeo studies will increase the number of patients with advanced cancer that have comprehensive genotyping of their cancer. The PRECISION 2 project consists of investigator-initiated phase II studies aiming to provide access to a targeted drug for patients whose tumors harbor actionable mutations in case the matched drug is not available through reimbursement or clinical trials in Belgium.

Guidelines for an optimal management of a malignant colorectal polyp. What is essential in a pathology report ?

Colorectal cancer (CRC) has become the most common malignancy in our country. Routine screening colonoscopy is on the rise. With the recent advances in endoscopic treatment, many T1 colorectal carcinomas are now found and their percentage amenable to endoscopic resection has increased. Endoscopists and pathologists dealing with the steadily increasing number of excised colorectal polyps have to collaborate closely to optimize patient care. Therapeutic management of patients after endoscopic resection is based on precise histological criteria that determine the risk of metastasis and the need for complementary surgery. This paper summarizes the procedures for the macroscopic management of endoscopic excisions and presents the identified risk factors which should be included in a standardized pathology report.

The choice of recipient does not have a bearing on early outcome in liver transplant patients receiving grafts from non-heart-beating donors: a reappraisal?

INTRODUCTION: Donation after cardiac death has reemerged as a potential way of increasing the supply of organs for transplantation. We retrospectively reviewed the outcomes of non-heart-beating donor (NHBD) liver transplantation (OLT) experience and compared with standard heart-beating donation (HBD) at a single center. METHODS: From October 2003 to November 2006, 13/111 liver transplantations were performed in our institution with NHBD. Living donor liver transplantation, splitting procedures, combined, and pediatric liver transplantations were excluded from this analysis. RESULTS: Donor population was similar in both groups. The median warm ischemia time was 10 minutes (range 6 to 38). The median cold ischemia times 6 hours and 16 minutes (2.4 to 6.30 hours and 9 hours and 14 minutes (2.15 to 15.35 hours) for NHBD and HBD groups, respectively (P = .0002). In the NHBD groups, 4/13 (31%) grafts were retransplanted within 3 months, due to ischemic biliary lesions with severe cholestasis (n = 3) or due to the occurrence of primary nonfunction (n = 1). The retransplantation rate was significantly lower in the HBD group (11/98, 11%; P = .03). One-year patient and graft survivals were 62% and 54% versus 86% and 79%, respectively, for the NHBD and HBD groups (P = .107 and P = .003). CONCLUSION: Liver grafts procured from donors after cardiac death accounted for a significantly greater retransplantation rates, mainly due to nonanastomotic biliary strictures. This risk must be taken into account when transplanting such grafts. Based upon this experience, NHBD cannot rival HBD to be a comparable source of quality organs for liver transplantation.

Rapid blood pressure control with minoxidil: acute and chronic effects on blood pressure, sodium excretion, and the renin-aldosterone system.

Changes in blood pressure, heart rate, electrolyte excretion, and the renin-angiotensin-aldosterone system were monitored before and after minoxidil was added to a regimen of a diuretic and propranolol hydrochloride in 12 severely hypertensive patients. None required more than 40 mg of minoxidil daily for control. On a constant intake, urinary sodium excretion decreased, while urinary potassium excretion remained stable. Heart rate, body weight, and plasma volume increased, while creatinine clearance did not change. Although plasma renin activity increased fourfold, the plasma aldosterone concentration did not increase. Six subjects were restudied after two months of minoxidil treatment. Although blood pressure control continued to be excellent in these subjects, plasma renin values and plasma volume had returned to pretreatment levels. These studies suggest that minoxidil rapidly and effectively lowers blood pressure. Although sodium retention accompanies minoxidil administration acutely, the effect is independent of aldosterone and may be transient.

A suspicious pancreatic mass in chronic pancreatitis: pancreatic actinomycosis.

Introduction. Pancreatic actinomycosis is a chronic infection of the pancreas caused by the suppurative Gram-positive bacterium Actinomyces. It has mostly been described in patients following repeated main pancreatic duct stenting in the context of chronic pancreatitis or following pancreatic surgery. This type of pancreatitis is often erroneously interpreted as pancreatic malignancy due to the specific invasive characteristics of Actinomyces. Case. A 64-year-old male with a history of chronic pancreatitis and repeated main pancreatic duct stenting presented with weight loss, fever, night sweats, and abdominal pain. CT imaging revealed a mass in the pancreatic tail, invading the surrounding tissue and resulting in splenic vein thrombosis. Resectable pancreatic cancer was suspected, and pancreatic tail resection was performed. Postoperative findings revealed pancreatic actinomycosis instead of neoplasia. Conclusion. Pancreatic actinomycosis is a rare type of infectious pancreatitis that should be included in the differential diagnosis when a pancreatic mass is discovered in a patient with chronic pancreatitis and prior main pancreatic duct stenting. Our case emphasizes the importance of pursuing a histomorphological confirmation.

Critical effects on catalytic function produced by amino acid substitutions at Asp804 and Asp808 of the alpha1 isoform of Na,K-ATPase.

At two intramembrane carboxyl-containing amino acids of the sheep alpha1 isoform of Na,K-ATPase (Asp804 and Asp808), both charge-conserving (Asp to Glu) and charge-deleting (Asp to Asn, Leu and Ala) replacements were made and the altered enzymes studied. Nucleotide changes encoding the amino acid substitutions were placed in a cDNA encoding a ouabain-resistant enzyme (sheep alpha1 RD) and the encoded enzymes were expressed in ouabain-sensitive HeLa cells. Transfections with cDNAs carrying all Asp804 substitutions, along with those carrying Asp808Ala, Asp808Asn, and Asp808Leu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Asp804 and Asp808. Only the expression of the Asp808Glu enzyme produced ouabain-resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Asp804Ala and Asp808Ala were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain-sensitive wild-type sheep alpha1 Na,K-ATPase, which was expressed in ouabain-resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity (Kd = 1-30 nM), indicating that the inactive proteins were synthesized and folded properly in the plasma membrane. The results demonstrate that carboxyl side chains at positions 804 and 808 are critical for enzyme catalytic function.

Cardiac hypertrophy and cardiac renin-angiotensin system in Dahl rats on high salt intake.

OBJECTIVE: On high salt intake, Dahl salt-sensitive rats develop cardiac hypertrophy disproportionate to the degree of hypertension. In the present studies, we assessed whether the cardiac hypertrophy induced by high salt depends on the development of hypertension per se, and leads to over-activity of the cardiac renin-angiotensin system (RAS). METHODS: Cardiac angiotensin converting enzyme (ACE) mRNA and activity, cardiac and plasma angiotensin I and II (AngI, II), as well as plasma renin activity (PRA) were assessed in Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats on high (1370 micromol/g food) or regular salt (120 micromol/g food) diet for 2-5 weeks. Cardiac ACE and hypertrophic response in Dahl S on high salt were also assessed after central blockade of sympathetic hyperactivity and hypertension. RESULTS: In Dahl S rats, ACE mRNA and activity of the left ventricle (LV) increased markedly after 4-5 weeks of high salt diet compared with Dahl S on the control diet and Dahl R on either diet Chronic intra-cerebroventricular treatment with Fab fragments blocking brain 'ouabain' prevented the hypertension by high salt in Dahl S rats but did not affect the salt-induced increases in LV weight or in LV ACE mRNA and activity. On regular salt diet, Dahl S rats demonstrated significantly lower cardiac AngI and AngII than Dahl R rats. However, high salt intake did not cause significant changes in cardiac AngI and II in either strain. On regular salt diet, PRA, plasma AngI and II were all significantly lower in Dahl S versus R. In Dahl S rats, high salt did not cause further decreases of the already low PRA or plasma AngI and II. CONCLUSIONS: These data indicate a low activity of both circulatory and cardiac RAS in Dahl S versus R rats. The marked cardiac hypertrophy and increase in cardiac ACE mRNA and activity induced by high salt in Dahl S do not depend on the increase in blood pressure. High salt intake did not increase cardiac AngII in Dahl S, suggesting that the increase in ACE mRNA and activity may be relevant for non-angiotensinergic mechanisms involved in cardiac hypertrophy.

High salt intake and the brain renin--angiotensin system in Dahl salt-sensitive rats.

OBJECTIVES: To assess changes in the activity of the brain renin-angiotensin system during (i) the development of salt-sensitive hypertension; and (ii) the prevention of salt-sensitive hypertension by blocking brain 'ouabain'. METHODS: In protocol I, angiotensin converting enzyme (ACE) mRNA and activity and angiotensin I and II levels were assessed in the hypothalamus and pons of Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats on regular (120 micromol Na+ per g) or high (1370 micromol Na+ per g) salt diet from 4-6 weeks or 4-9 weeks of age. In protocol II, ACE mRNA and activity were assessed in the hypothalamus and pons in Dahl S on regular or high salt treated with intracerebroventricular (i.c.v.) Fab fragments blocking brain 'ouabain' or gamma-globulins, and in Dahl R on high or regular salt ACE mRNA was assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay and angiotensin I and II by radioimmunoassay after high-performance liquid chromatography. In protocol III, effects of i.c.v. angiotensin I and i.c.v. bradykinin on renal sympathetic nerve activity (RSNA), heart rate and blood pressure before and after i.c.v. captopril were assessed in Dahl S and R rats on regular or high salt intake from 4-8 weeks of age. RESULTS: High salt diet caused a gradual, but marked increase in blood pressure in Dahl S but not Dahl R rats. Dahl S rats showed small but significant increases in ACE mRNA in the hypothalamus on regular salt diet. In Dahl S rats on high salt diet for 2 or 5 weeks ACE mRNA levels significantly increased in both hypothalamus and pons, compared with Dahl R rats on either diet or Dahl S rats on regular diet. After 5 weeks of high salt diet, ACE mRNA levels in the hypothalamus in Dahl S rats were almost three-fold higher and in the pons two-fold higher than in Dahl R rats on either diet or Dahl S on regular salt diet. High salt diet also increased ACE activity of the hypothalamus and pons in Dahl S but not Dahl R. Consistent with this increased ACE activity, central responses to angiotensin I were clearly enhanced and to bradykinin markedly diminished in Dahl S on high salt intake. Chronic blockade of brain 'ouabain' by i.c.v. Fab fragments prevented the increases in blood pressure, ACE mRNA and activity in the hypothalamus and pons by high salt intake in Dahl S rats. Angiotensin I levels in the hypothalamus and pons were similar in both groups of rats and there were no significant changes caused by high salt diet in Dahl S and R rats. On regular salt intake angiotensin II levels in the hypothalamus of Dahl S rats showed a significant decrease as compared with Dahl R rats on regular salt diet, and were similar in the pons of the two strains. High salt intake did not affect angiotensin II levels in either hypothalamus or pons in Dahl S and R rats. CONCLUSIONS: These results indicate that high salt intake increases blood pressure, ACE expression and activity in the hypothalamus and pons of Dahl S rats without a parallel increase in angiotensin II levels. Effects of high salt intake on ACE mRNA and activity appear to be secondary to activation of brain 'ouabain'.

Cation and cardiac glycoside binding sites of the Na,K-ATPase.

From the structural data obtained by systematically altering residues of the Na,K-ATPase, we are beginning to understand portions of how this active cation transporter couples hydrolysis of ATP with the vectorial movement of cations against their ionic gradients. In addition, the inhibitory action of cardiac glycosides and their interaction sites on the protein has focused our attentions on a catalytic core of the protein involving the H5-H6 transmembrane segment. In future investigations, both the ATP and the Na+ sites of the Na,K-ATPase must be uncovered to refine the structural picture of this complex transporter.

Structure-function studies of the Na,K-ATPase.

Na,K-ATPase catalyzes the movement of sodium and potassium ions across the cell membrane utilizing ATP as an energy source. This enzyme is present in almost all tissues of higher organisms but is most abundant in the kidney where it is responsible for reabsorbing sodium ions from the glomerular filtrate. The enzyme is composed of two subunits and serves as the receptor for cardiac glycosides. Utilizing an expression/selection system it has been possible to identify several amino acid residues that affect sensitivity to the cardiac glycoside, ouabain. Those identified to date are located in the first transmembrane region and first extracellular region. The fact that amino acid residues within a transmembrane region affect ouabain sensitivity suggests that the drug is partially internalized in the lipid bilayer. In an effort to determine whether any of the amino acid residues which affect ouabain sensitivity interact with the sugar part of cardiac glycosides, ouabain and ouabagenin were tested in terms of their ability to inhibit enzyme containing substitutions at these positions. The two compounds differ in that ouabagenin lacks a sugar moiety. Interestingly, the ratio of I50's for the substituted enzymes remains the same as the wild type, which suggests that the amino acids identified as determinants of ouabain sensitivity to date are not likely to interact with the sugar. Another set of studies focused on cation binding. It has been proposed that cation transport involves negatively charged residues in one or more transmembrane regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disorder. Systemic treatment of IBD patients with anti-tumor necrosis factor (TNF)-alpha antibodies has proven to be a highly promising approach, but several drawbacks remain, including side effects related to systemic administration and high cost of treatment. Lactococcus lactis was engineered to secrete monovalent and bivalent murine (m)TNF-neutralizing Nanobodies as therapeutic proteins. These therapeutic proteins are derived from fragments of heavy-chain camelid antibodies and are more stable than conventional antibodies. L. lactis-secreted anti-mTNF Nanobodies neutralized mTNF in vitro. Daily oral administration of Nanobody-secreting L. lactis resulted in local delivery of anti-mTNF Nanobodies at the colon and significantly reduced inflammation in mice with dextran sulfate sodium (DSS)-induced chronic colitis. In addition, this approach was also successful in improving established enterocolitis in interleukin 10 (IL10)(-/-) mice. Finally, L. lactis-secreted anti-mTNF Nanobodies did not interfere with systemic Salmonella infection in colitic IL10(-/-) mice.In conclusion, this report details a new therapeutic approach for treatment of chronic colitis, involving in situ secretion of anti-mTNF Nanobodies by orally administered L. lactis bacteria. Therapeutic application of these engineered bacteria could eventually lead to more effective and safer management of IBD in humans.

Colon mucosa of patients both with spondyloarthritis and Crohn's disease is enriched with macrophages expressing the scavenger receptor CD163.

BACKGROUND: Crohn's disease is associated with an increased number of macrophages in ileal and colonic mucosa. Data on macrophages in gut mucosa of patients with spondyloarthritis (SpA) are scarce. OBJECTIVE: To investigate macrophages and other antigen presenting cells in gut mucosa from patients with SpA and Crohn's disease, given the relationship between both entities. METHODS: Biopsy specimens from patients with SpA, Crohn's disease, ulcerative colitis, and from controls were immunohistochemically stained with different markers for macrophages and dendritic cells. Slides were scored semiquantitatively on a four point scale. RESULTS: SpA and Crohn's disease were associated with large numbers of CD68+ macrophages. Colon mucosa of both patients with SpA and Crohn's disease, but not ulcerative colitis, showed increased numbers of macrophages expressing the scavenger receptor CD163. CONCLUSIONS: Macrophages expressing the scavenger receptor CD163 are increased in colonic mucosa in SpA and in Crohn's disease, highlighting the relationship between these entities. The increased number of CD163+ macrophages in colon mucosa of patients with SpA suggests this is another argument for a role of macrophage scavenger receptors in this group of diseases.

Central nervous system norepinephrine release during hypotension and hyperosmolality in conscious rats.

Extracellular norepinephrine (NE) levels in the paraventricular/anterior hypothalamic area (P/A) and in the dorsomedial medulla (DM) in conscious Sprague-Dawley rats were estimated by in vivo microdialysis before, during, and after sustained hypotension (75 mmHg mean arterial pressure) produced either by hemorrhage (Hem) or by 2-chloroadenosine infusion (2-Cl-ADO, 2.6-26.0 micrograms/min iv). P/A and DM NE were also measured before, during, and after hypertonic saline infusion (HTS; 1.5 M NaCl at 10 microliters.100 g-1.min-1 iv). P/A and DM NE increased during both Hem and 2-Cl-ADO and returned to baseline after reinfusion of hemorrhaged blood or after 2-Cl-ADO was stopped. However, Hem caused greater increases in P/A NE than 2-Cl-ADO despite equivalent decreases in blood pressure. Hem and 2-Cl-ADO produced equivalent changes in DM NE. HTS did not change P/A or DM NE despite increases in blood pressure of approximately 15 mmHg and plasma osmolality of approximately 30 mosmol/kgH2O. We conclude that 1) hypotension increases P/A and DM NE, which may mediate compensatory responses, 2) Hem is a more potent stimulus for NE release in the P/A than isovolemic hypotension induced by 2-Cl-ADO, and 3) the hypertensive response to HTS does not involve changes in P/A or DM NE.

Nonpolar amino acid substitutions of potential cation binding residues glu-955 and glu-956 of the rat alpha 1 isoform of Na+, K(+)-ATPase.

Side chain oxygens are critical in the binding of cations by several macromolecules, and chemical labeling studies suggest that the carboxyl oxygens of E953 of the pig kidney Na, K-ATPase are essential for Na+ and K+ ligation. An adjacent residue, E954, is highly conserved and may also be important in cation binding. Replacement of the corresponding glutamates in the rat alpha 1 isoform (E955 and E956) with glutamines or aspartic acids has only a modest effect on enzyme activity, but both substituted amino acids contain at least one side chain oxygen, and may fulfill the role of the wild-type glutamates in binding cations. Since substitutions of amino acids lacking an oxygenated side chain have not been made at positions 955 and 956, nonpolar amino acid replacements (E955A, E956A, E955A-E956A, and E955L-E956L) were made, and the effects of the substitutions on the cation dependence properties of the mutant enzymes were examined. The substitutions were made using site-directed mutagenesis of the rat alpha 1 cDNA (which encodes a ouabain-resistant alpha subunit), followed by transfection of wild-type and mutant cDNAs into ouabain-sensitive HeLa cells, with subsequent expression of the altered proteins. Transfection with all cDNA constructs resulted in the ouabain resistance of transfected HeLa cells, demonstrating that the modified Na, K-ATPase in each case was functional. Ouabain resistance of Na, K-ATPase activity was increased 1,000-fold in microsomal membranes isolated from cells transfected with wild-type rat alpha 1 (WT) and all mutant cDNA constructs, compared to activity in membranes prepared from untransfected HeLa cells. This confirmed the expression of a ouabain-resistant enzyme in all transfected cell lines, and the enhanced ouabain resistance permitted the study of the exogenous, expressed Na, K-ATPase in the presence of 5.0 microM ouabain. Cation stimulation of exogenous Na, K-ATPase activity was not affected by the E955A substitution and only slightly by the E956A replacement (K0.5(Na+) of 3.40 +/- 0.21, 3.30 +/- 0.22, and 6.60 +/- 0.55 mM NaCl for WT, E955A and E956A, respectively; K0.5(K+) of 0.78 +/- 0.01, 0.74 +/- 0.10, and 0.56 +/- 0.11 mM KCl). Even doubly substituted enzymes had only mild alterations in cation dependence properties (K0.5(Na+) of 7.54 +/- 0.23, 7.14 +/- 0.10 mM NaCl for E955A-E956A, E955L-E956L, respectively; K0.5(K+) of 0.43 +/- 0.13, 1.83 +/- 0.13 mM KCl). The results demonstrate that there are no requirements for an oxygenated side chain at positions 955 and 956 for normal or nearly normal cation dependence.

Central nervous system catecholamine content and norepinephrine release after AV3V ablation.

Electrolytic ablation of the periventricular tissue surrounding the anteroventral third cerebral ventricle (AV3V-X) results in immediate increases in norepinephrine release in the paraventricular hypothalamic nucleus/anterior hypothalamus (P/A) and in the dorsomedial medulla (DM) and depletion of norepinephrine and epinephrine throughout the brain. The present study determined whether catecholamine depletion and/or altered norepinephrine release persisted after recovery from the acute effects of AV3V-X. Fourteen to eighteen days after surgery, catecholamine concentrations throughout the brain and norepinephrine release in the P/A and DM during hemorrhage were measured in control-operated and AV3V-X rats. AV3V-X decreased epinephrine content only in the midbrain, increased norepinephrine only in the cortex, and produced no differences in central nervous system dopamine. Hemorrhage-induced norepinephrine release was significantly greater in the P/A of AV3V-X rats than in control animals, whereas norepinephrine release in the DM increased equally in both groups. Thus AV3V-X does not result in chronic global central nervous system catecholamine depletion and enhances norepinephrine release in the P/A during hemorrhage.

Axon destruction and adrenergic systems mediate pressor responses after AV3V lesions.

These studies investigated the neural tissue and peripheral mechanism mediating the transient pressor response following electrolytic ablation of the periventricular tissue surrounding the anteroventral third ventricle (AV3V) of the rat. Arterial blood pressure was monitored in conscious animals for 2 h following either microinjection of kainic acid (AV3V-KA) or electrolytic lesions (AV3V-X) in the AV3V region or control procedures (Cont). Blood pressure did not change in AV3V-KA (4 +/- 3 mmHg) or Cont rats but significantly increased in AV3V-X animals (20 +/- 4 mmHg). The pressor response following AV3V-X was not altered by pretreatment with MK-422 (converting-enzyme inhibitor), TMe-AVP (vasopressin antagonist), or hexamethonium (ganglionic blocker). However, intravenous administration of prazosin (alpha-adrenergic blocker) or bilateral adrenalectomy abolished the increase in blood pressure. Furthermore, plasma concentrations of norepinephrine were significantly higher in AV3V-X rats (1,125 +/- 150 pg/ml) compared with Cont animals (322 +/- 83 pg/ml) following treatment. These data indicate that the acute hypertensive response following AV3V-X is caused by the destruction of fibers of passage and results from circulating catecholamines of adrenal origin.

Hypertension and alterations in central catecholamines after preoptic recess lesions.

Catecholamine concentrations were measured in brain regions of rats with lesions of the anteroventral third ventricle (AV3V-X rats) or parietal cortex (CORT-X) and of control-operated (CONT) rats. Three hours after surgery, total norepinephrine (NE) concentrations in all brain regions measured of AV3V-X rats were less than those of CONT or CORT-X rats. Epinephrine (EPI) concentrations were decreased in the medulla and hypothalamus, but dopamine levels were unchanged. Also, extracellular NE concentration, estimated by in vivo microdialysis techniques, was increased in the anterior hypothalamic nucleus (AHNE) and dorsomedial medulla (DMMNE) of AV3V-X rats compared with CONT rats. Increased DMMNE paralleled blood pressure increases. DMMNE did not change after blood pressure was increased by intravenous angiotensin II infusion (60 ng/min) in CONT rats. Thus 1) AV3V lesions result in decreased NE and EPI concentrations in multiple brain regions; 2) acute reductions in central NE in AV3V-X rats are associated with increased extracellular NE in the AH and DMM, suggesting increased NE release; and 3) although increased DMMNE is temporally related to increased blood pressure following AV3V lesions, increased DMMNE is not a response to the hypertension per se.

Role of endogenous brain "ouabain" in the sympathoexcitatory and pressor effects of sodium.

Endogenous cardiac glycoside inhibitors of the Na, K-ATPase (called "ouabain" here) with structures similar to plant ouabain have been isolated in several tissues, including the adrenal cortex and the brain. Recent studies have demonstrated that "ouabain" in the anteroventral third ventricle (AV3V) region of the hypothalamus mediates the sympathoexcitatory and pressor responses to a high sodium diet in Dahl salt-sensitive (Dahl-S) and spontaneously hypertensive (SHR) rats. Although the mechanisms regulating the biosynthesis, release and deactivation of CNS "ouabain" remain unknown, the discovery of the importance of brain "ouabain" in cardiovascular regulation creates a novel path for the development of antihypertensive pharmacopeia.

Site-directed mutagenesis of a predicted cation binding site of Na, K-ATPase.

Chemical modification and proteolytic digestion studies have identified a transmembrane glutamic acid residue (E953) of the alpha subunit of the pig kidney Na, K-ATPase as a possible cation binding site [Goldshleger et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6911-6915]. In addition, an adjacent glutamate (E954) is conserved in all species and isoforms and may also be involved in cation binding. To further explore the role of these residues in ion transport, we have utilized a mutagenesis-expression strategy. This approach avoids the introduction of a large chemical moiety into the protein and allows specific amino acid substitutions to be introduced. Glutamic acid residues 955 and 956 of the rat alpha-1 subunit (corresponding to glutamates 953 and 954 of the pig kidney Na, K-ATPase) were replaced separately and together using site-directed mutagenesis of the rat alpha-1 cDNA. The mutant cDNAs were expressed in ouabain-sensitive HeLa cells. This system makes it possible to rapidly identify amino acid substitutions which significantly impair enzyme function, as substitutions which do not affect enzyme activity will yield colonies in the presence of ouabain, while substitutions which severely impair function will prevent or limit growth of the ouabain-sensitive HeLa cells. The amino acid replacements (E955Q, E956Q, E955Q-E956Q, E955D-E956D) all resulted in the growth of ouabain-sensitive cells, demonstrating that the modified Na, K-ATPase in each case was functional. To further study the altered enzymes, ouabain-resistant colonies were isolated and expanded into stable cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo renal production and tubular secretion of tyramine.

Para-tyramine (p-TYM) is a predominant urinary amine in humans, rabbit, rat and dog, and its urinary excretion rate may reflect central nervous system pathophysiology. However, the source of urinary p-TYM is not known, nor have the mechanisms regulating its excretion been characterized. The present study, by using renal clearance techniques, examined the sources of urinary p-TYM and the mechanism of excretion in anesthetized rabbits. In all studies, the renal clearance of p-TYM was compared with that of norepinephrine (NE). Base-line delivery to the kidney of p-TYM in plasma was 8.6 +/- 1.6 ng/min (mean +/- S.E.M., n = 16), whereas the mean urinary excretion rate of p-TYM was 26.5 +/- 3.6 ng/min during the same period (P < .001 vs. delivery). In three separate series of experiments, either vehicle (n = 5) or a specific inhibitor of renal tubular organic cation secretion, cyanine 863 (6 mg/kg, n = 7), or a specific inhibitor of aromatic-amino acid-decarboxylase, alpha-mono-fluoromethyldopa (FMD, 4 mg/kg, n = 5), were infused i.v., Mean arterial pressure, glomerular filtration rate, renal plasma flow and urine flow rate were unchanged in all studies. The renal clearances of p-TYM (Cp-TYM) and NE (CNE) were unchanged only after vehicle. After cyanine 863, Cp-TYM was decreased to 36% of control (P < .01), whereas CNE decreased to 21% of its base-line value (P < .01). After FMD Cp-TYM was reduced to 2% of control (P < .05), whereas CNE decreased by 44% (P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)