Biomedical subjects

J D Altman

Publications and source records attributed to J D Altman.

83 records · Page 5Linked to original sources

PubMed1995

Effect of beta-adrenergic receptor blockade on blood flow to collateral-dependent myocardium during exercise.

BACKGROUND: beta-Adrenergic receptors have been identified in isolated coronary collateral blood vessels, but their functional significance in the intact heart has not been demonstrated. METHODS AND RESULTS: We measured myocardial blood flow with radioactive microspheres in normal and collateral-dependent myocardium in eight dogs trained to run on a treadmill before and after beta-adrenergic blockade with propranolol, 200 micrograms/kg, a dose that effectively inhibited the increase in coronary blood flow produced by selective beta 1- and beta 2-adrenergic agonists. Collateral vessel growth was stimulated with 2-minute intermittent occlusions of the left anterior descending artery followed by permanent occlusion. During control exercise, blood flow in the collateral zone was 38 +/- 5% less than in the normal zone. At identical levels of exercise, with heart rate maintained constant by atrial pacing, propranolol decreased mean blood flow in the collateralized myocardium from 1.93 +/- 0.17 to 1.50 +/- 0.14 mL.min-1.g-1 (P < .01), while increasing the subendocardial to subepicardial blood flow ratio from 0.78 +/- 0.11 to 0.91 +/- 0.10 (P < .05). The decrease in collateral zone blood flow in response to propranolol resulted from an increase in both transcollateral resistance from 25.9 +/- 2.3 to 35.2 +/- 4.3 mm Hg.mL-1.min.g (P < .05) and small-vessel resistance in the collateral-dependent myocardium from 30.9 +/- 4.7 to 44.0 +/- 8.8 mm Hg.mL-1.min.g (P < .07). Blood flow to the normal zone was also significantly reduced from 3.14 +/- 0.21 to 2.23 +/- 0.12 mL.min-1.g-1 (P < .01) after propranolol. CONCLUSIONS: beta-Adrenergic blockade decreased blood flow to collateral-dependent myocardium during exercise. These results indicate that beta-adrenergic receptor activation contributes to vasodilation of coronary collateral vessels during exercise.

Animals↗

PubMed1995

Cyclooxygenase blockade limits blood flow to collateral-dependent myocardium during exercise.

Cyclooxygenase blockade has been found to cause vasoconstriction of coronary collateral vessels in open-chest animals. This study was carried out to determine whether cyclooxygenase blockade can limit blood flow to collateral-dependent myocardium during exercise. Studies were performed in 8 adult mongrel dogs in which intermittent followed by permanent occlusion of the left anterior descending coronary artery produced an area of collateral-dependent myocardium. Myocardial blood flow was measured with radioactive microspheres at rest and during treadmill exercise to produce heart rates of 215 +/-0 7 beats/min. At rest collateral zone blood flow (1.00 +/- 0.10 ml/min per g) was significantly less than normal zone flow (1.23 +/- 0.14) (P < 0.05). During control exercise blood flow increased 91 +/- 22% in the collateral zone and 102 +/- 28% in the normal zone (each P < 0.05). Thirty minutes after cyclooxygenase blockade with indomethacin (5 mg/kg i.v.) blood flow in the normal zone and the collateral zone was not different from control during resting conditions. Indomethacin did not change heart rate or arterial pressure during exercise, but significantly increased the aortic-to-distal coronary pressure gradient from 33 +/- 3 to 40 +/- 5 mmHg (P < 0.05). Indomethacin increased transcollateral resistance during exercise by 42 +/- 10% (P < 0.05); this was associated with a 27 +/- 11% decrease in subendocardial flow in the collateral zone (P < 0.05) with no significant change in subepicardial flow, and no change in normal zone blood flow. These findings demonstrate that in the intact awake animal cyclooxygenase blockade causes coronary collateral vasoconstriction which can impair blood flow to the dependent myocardium during exercise.

Animals↗

PubMed1994

Effect of inhibition of nitric oxide formation on coronary blood flow during exercise in the dog.

OBJECTIVE: The aim was to test the hypothesis that nitric oxide (or a related compound) contributes to the coronary vasodilatation during physiological increases of myocardial O2 consumption that occur with exercise. METHODS: Active hyperaemia associated with graded treadmill exercise and coronary reactive hyperaemia were examined in chronically instrumented awake dogs during control conditions and after administration of the nitric oxide synthase inhibitor, N-nitro-L-arginine (LNNA). RESULTS: LNNA blunted the response to intracoronary acetylcholine, with an 80(SEM 6)% decrease in the maximum acetylcholine induced coronary vasodilatation, but did not alter the response to sodium nitroprusside. Increases of myocardial oxygen requirements during treadmill exercise were associated with progressive increases of coronary blood flow. LNNA caused a significant increase in arterial pressure at rest and during exercise, and this was associated with slightly but significantly higher myocardial oxygen consumption. Coronary blood flow-during exercise was also slightly higher after LNNA, while coronary vascular resistance was unchanged. Coronary sinus PO2 was slightly but significantly lower during exercise after LNNA, indicating that coronary vasodilatation in response to the increased myocardial oxygen demands during exercise was slightly blunted by LNNA. LNNA did not alter the peak increase in blood flow during reactive hyperaemia following a 15 s coronary occlusion, but decreased the duration of the response and decreased reactive hyperaemia debt repayment from 300(56)% during control conditions to 182(36)% after LNNA (p < 0.01). CONCLUSIONS: LNNA antagonised coronary vasodilatation in response to acetylcholine and blunted coronary reactive hyperaemia, but did not substantially impair the coronary vasodilatation associated with increased myocardial oxygen requirements produced by exercise. These findings fail to support an essential role for nitric oxide in coronary resistance vessel dilatation during exercise in the dog.

Acetylcholine↗

PubMed1994

The mechanism of coronary collateral vasoconstriction in response to cyclooxygenase blockade.

The present study was performed to examine the mechanism by which cyclooxygenase blockade produces vasoconstriction in well-developed coronary collateral vessels. Eight dogs were studied 4 to 6 months after occlusion of the left anterior descending coronary artery (LAD) had been performed to stimulate collateral vessel growth. At the time of study, the LAD was cannulated at the site of occlusion for measurement of retrograde blood flow as an index of collateral blood flow. Levels of 6-ketoprostaglandin F1 alpha were 32 +/- 13% higher in blood diverted from the collateral-dependent LAD than in aortic blood (P < .05); the increase in this stable product of prostacyclin metabolism indicated production of prostacyclin across the coronary collateral system. Administration of arachidonic acid into the left main coronary artery to reach collateral vessels entering the LAD resulted in a 21 +/- 6% increase in retrograde flow (P < .01), demonstrating cyclooxygenase activity with production of vasodilator prostaglandins in the collateral system. Ibuprofen (10 mg/kg IV) caused a 55 +/- 7% decrease in retrograde flow (P < .03), suggesting that cyclooxygenase blockade inhibited tonic production of vasodilator prostaglandins in the collateral system. In contrast, neither thromboxane synthase inhibition with dazmegrel nor thromboxane receptor blockade with SQ 30741 caused a significant change in collateral flow, thus failing to support thromboxane-induced collateral constriction. After cyclooxygenase blockade, prostacyclin infused into the left main coronary artery was able to restore retrograde flow to the preibuprofen level.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗

PubMed1993

Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli.

Class II major histocompatibility complex molecules play a major role in the immune response by binding peptide fragments of exogenous antigens and displaying them on the surfaces of antigen-presenting cells, where they can be recognized by T cells. To facilitate structural and functional studies of these molecules, we have produced truncated alpha and beta chains of the murine class II molecule I-Ek in Escherichia coli (Ec-I-Ek) and have developed conditions to fold them in the presence of specific peptides with yields of complex approaching 2%. Reconstitution is specific since only unlabeled peptide known to bind I-Ek compete with biotinylated peptide, as assessed by ELISA. Complexes of the refolded heterodimer (Ec-I-Ek) with either of two different peptide antigens remain associated during nonreducing SDS/PAGE. Immobilized Ec-I-Ek-peptide complexes stimulate lymphokine production by three T-cell clones in an antigen-specific manner with a dose-response relation comparable to previously described soluble I-Ek molecules produced in CHO cells. These results demonstrate that folding of Ek alpha and Ek beta polypeptides does not require any other protein to produce the biologically relevant conformation and that carbohydrate modification of this class II molecule is not necessary for alpha beta T-cell recognition.

Amino Acid Sequence↗

PubMed1993

Two-dimensional nuclear magnetic resonance analysis of a labeled peptide bound to a class II major histocompatibility complex molecule.

The formation of peptide/major histocompatibility complex (MHC) complexes and their subsequent recognition by T cells is a pivotal event in the initiation of an immune response. While X-ray crystal structures are now available for class I MHC/peptide complexes, little detailed structural information is known about the class II MHC equivalent, and there are no solution structure data for either. A 16 amino acid residue moth cytochrome c peptide (residues 88 to 103) was 13C-labeled for two-dimensional isotope-edited NMR analysis. The peptide was labeled either selectively in the methyl groups of alanine residues or uniformly at every carbon position, and bound to unlabeled soluble mouse I-Ek class II MHC molecules. Although alpha-helical in the native cytochrome c protein and with no uniform structure in solution, the peptide is bound to the I-Ek molecule with the alpha-carbon atoms of the 11 C-terminal residues held in the binding groove. This indicates that the class II MHC peptide binding site is somewhat larger than that of class I MHC molecules (> or = 11 amino acid residues versus 8 to 10 amino acid residues), consistent with recent data on eluted peptides. Despite the large size of the complex (approximately 70 kDa), nuclear Overhauser effects are clearly detectable between peptide side-chains and the MHC molecule. Indications of the buried or exposed nature of particular side-chains within the bound peptide are derived from the NMR data and these are used together with information from previous biological studies to propose a crude model of the interaction of the peptide with the groove of the MHC molecule. We find no evidence for a conformational change in the peptide/MHC complex in the spectra at pH 5.0 versus pH 7.0, despite a 40-fold faster on-rate for the peptide at the lower pH value.

Amino Acid Sequence↗

PubMed1993

Effect of aspirin on coronary collateral blood flow.

BACKGROUND: Although aspirin exerts beneficial antiplatelet activity in patients with coronary artery disease, cyclooxygenase blockade produced by aspirin causes a potentially deleterious effect by interrupting endothelial production of prostacyclin. Collateral vessels that develop in response to coronary occlusion display prominent endothelial cell proliferation and undergo vasoconstriction in response to indomethacin. This study was performed to test the hypothesis that cyclooxygenase blockade with aspirin would cause constriction of coronary collateral vessels and that such vasoconstriction would be reversed with nitroglycerin. METHODS AND RESULTS: Collateral vessel growth was induced by embolic occlusion of the left anterior descending coronary artery in dogs. Four to 6 months later, coronary collateral flow was measured as retrograde flow from the cannulated collateral-dependent artery. Aspirin (1 mg/kg i.v.) caused 70 +/- 8% blockade of the increase in coronary blood flow produced by intra-arterial arachidonic acid and decreased retrograde flow from 37 +/- 7 to 28 +/- 7 ml/min (p < 0.03). Increasing the dose of aspirin to 15 mg/kg i.v. caused 91 +/- 3% blockade of the response to arachidonic acid and further decreased retrograde flow to 21 +/- 4 ml/min (p < 0.01). After aspirin administration, nitroglycerin (150 micrograms/min i.c.) reversed the collateral constriction and increased retrograde flow to 37 +/- 10 ml/min (p < 0.01). CONCLUSIONS: These data suggest that products of cyclooxygenase metabolism cause tonic vasodilation of well-developed coronary collateral vessels. Blockade of cyclooxygenase with even low-dose aspirin caused collateral vessel constriction with a decrease in collateral blood flow. However, nitroglycerin was able to fully reverse aspirin-induced collateral vasoconstriction and restore flow to the control level.

Animals↗

PubMed1993

Role of K+ATP channels in coronary vasodilation during exercise.

BACKGROUND: The mechanism of metabolic regulation of coronary vascular tone is still unclear. Therefore, we examined the role of vascular smooth muscle K+ATP channels in regulating coronary blood flow under resting conditions, during increments in myocardial metabolic demand produced by treadmill exercise, and in response to a brief ischemic stimulus. METHODS AND RESULTS: Ten chronically instrumented dogs were studied at rest and during a four-stage exercise protocol under control conditions and during intracoronary infusion of the K+ATP channel blocker glibenclamide at rates of 10 and 50 micrograms.kg-1 x min-1. Glibenclamide (50 micrograms.kg-1 x min-1) decreased coronary blood flow at rest from 51 +/- 4 to 42 +/- 6 mL/min (P < .05), decreased myocardial oxygen consumption from 5.70 +/- 0.31 to 4.11 +/- 0.56 mL O2/min (P < .05), and decreased systolic wall thickening from 21 +/- 3% to 12 +/- 3% (P < .05). The depression of systolic wall thickening produced by glibenclamide was reversed when coronary blood flow was restored to the control level with intracoronary nitroprusside, indicating a primary effect of glibenclamide on coronary flow during resting conditions. However, glibenclamide did not impair the increases of coronary blood flow, myocardial oxygen consumption, and systolic wall thickening that occurred during exercise. In eight resting awake dogs, 50 micrograms.kg-1 x min-1 glibenclamide decreased the peak reactive hyperemia blood flow rate following a 20-second coronary occlusion from 149 +/- 14 mL/min during control conditions to 111 +/- 15 mL/min (P < .05), decreased the duration of reactive hyperemia from 49 +/- 6 to 33 +/- 3 seconds (P < .05), and decreased reactive hyperemia excess flow from 33 +/- 5 to 20 +/- 4 mL (P < .05). CONCLUSIONS: These data demonstrate that K+ATP channels modulate coronary vasomotor tone under resting conditions and contribute to coronary vasodilation during ischemia. However, the coronary vasculature retains the capacity to dilate in response to increases in oxygen demand produced by exercise when K+ATP channels are blocked.

Adenosine Triphosphate↗

PubMed1991

Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin.

The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy.

Amino Acid Sequence↗

PubMed1985

Development of an affinity chromatography resin for the purification of carcinogen binding proteins from mouse liver.

Pyrene, a structural analog of benzo[a]pyrene, is an effective competing ligand for high affinity carcinogen binding proteins in mouse liver. A pyrene-derivatized Sepharose gel was prepared for affinity chromatography purification of these proteins, and adsorbs all detectable [3H]B[a]P-binding activity from hepatic cytosol with the adsorption of less than 1% of total protein. Specific carcinogen binding activity is recovered from pyrene-derivatized Sepharose columns with the enrichment of a 33 kDa polypeptide. This chromatography resin represents a major step in the isolation of these unusual receptor-like binding proteins for aromatic hydrocarbon carcinogens.

Animals↗

PubMed1985

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line.

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF).

Aryl Hydrocarbon Hydroxylases↗