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Biomedical subjects

R M Graham

Publications and source records attributed to R M Graham.

At least 109 records · Page 6Linked to original sources
PubMed1988

Responsiveness of plasma atrial natriuretic factor to short-term changes in left atrial hemodynamics after percutaneous balloon mitral valvuloplasty.

To assess the effect of short-term alteration of left atrial pressure and volume on the circulating plasma level of atrial natriuretic factor, 11 patients with left atrial hypertension due to mitral stenosis were studied at the time of percutaneous balloon mitral valvuloplasty. Hemodynamic measurements and plasma atrial natriuretic factor levels were obtained before, immediately (5 to 10 min) after and 24 h after valvuloplasty, and echocardiographic left atrial size was determined before and 24 h after valvuloplasty. Immediately after valvuloplasty, left atrial pressure decreased from 28 +/- 2 to 10 +/- 1 mm Hg (p less than 0.0005), mitral pressure gradient decreased from 20 +/- 2 to 7 +/- 1 mm Hg (p less than 0.0005), mitral valve area increased from 0.8 +/- 0.1 to 1.9 +/- 0.2 cm2 (p less than 0.0005) and plasma atrial natriuretic factor level rose from 249 +/- 42 to 348 +/- 50 pg/ml (p less than 0.01). This short-term rise in atrial natriuretic factor level may reflect a transient increase in left atrial pressure associated with balloon occlusion of the mitral valve.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Alpha-adrenergic vasoconstriction and receptor subtypes in large coronary arteries of calves.

We investigated alpha-adrenoceptor subtype distribution in large coronary arteries from both functional and biochemical perspectives. The effects of intracoronary administration of the selective alpha 1-adrenoceptor agonist phenylephrine (0.1-5.0 micrograms/kg ic), of the selective alpha 2-adrenoceptor agonist B-HT 920 (0.1-5.0 micrograms/kg ic), and of the mixed alpha 1 + 2-adrenoceptor agonist norepinephrine (0.01-0.5 micrograms/kg ic) were examined on measurements of left circumflex coronary artery diameter in conscious calves. After beta-adrenergic blockade, equivalent reductions in large coronary artery diameter were observed with phenylephrine (-6.9 +/- 0.7%), B-HT (-5.9 +/- 0.5%), and norepinephrine (-6.0 +/- 0.4%). Phenylephrine-induced constrictions were abolished by prazosin, an alpha 1-selective antagonist, but unaffected by rauwolscine, an alpha 2-selective antagonist. Conversely, the B-HT-induced constriction was abolished by rauwolscine but unaffected by prazosin. Coronary constriction with norepinephrine was attenuated with either prazosin or rauwolscine and abolished by the two antagonists combined. Ligand-binding studies in which [3H]prazosin and [3H]rauwolscine and sarcolemmal membranes were used revealed an alpha 1-adrenoceptor density of 15 +/- 3.1 fmol/mg protein with a dissociation constant (KD) of 0.7 +/- 0.2 nM and an alpha 2-adrenoceptor density of 68 +/- 5.1 fmol/mg protein, with a KD of 7.4 +/- 1.2 nM. Thus large coronary arteries of the calf contain both alpha 1- and alpha 2-adrenoceptor subtypes, each of which elicits constriction of the large coronary artery in the conscious animal.

Adrenergic alpha-Agonists↗
PubMed1988

Direct effects of alpha 2-adrenergic receptor stimulation on intravascular systemic capacity in the dog.

The role of alpha 2-adrenergic receptor stimulation in the regulation of systemic vascular capacity and venous return, a major determinant of cardiac output, is not well understood. With the influence of the central nervous system isolated from the systemic circulation, the direct peripheral vascular effects of two specific, chemically distinct alpha 2-adrenergic receptor agonists, UK 14,304 and B-HT 920, were investigated in 19 dogs on total cardiopulmonary bypass with constant arterial perfusion and central venous pressure. Five-minute intra-arterial infusions of UK 14,304 (200 micrograms/min) resulted in increased arterial resistance (mean arterial pressure increased 18 +/- 4 [SEM] mm Hg; p less than 0.01) and a decrease in systemic vascular capacity (81 +/- 20 ml; p less than 0.01). This decrease in systemic vascular capacity appears to result from vasoconstriction, since there was no decrease in transhepatic resistance to portal flow and no significant change in hepatic vein flow to suggest redistribution of arterial blood flow. Yohimbine abolished both the arterial and systemic capacity effects, whereas prazosin did not. Intra-arterial administration of B-HT 920 (200 theta grams/min) in five dogs produced similar changes in arterial resistance and systemic capacity. These findings provide direct evidence for beta 2-adrenergic control, not only of arterial resistance but also of systemic vascular capacity, which in the intact animal would increase venous return to the heart.

Adrenergic alpha-Agonists↗
PubMed1988

Neurogenic histaminergic vasodilation in canine skeletal muscle: mediation by alpha 2-adrenoceptor stimulation.

This study examines the neurogenic effect of alpha 2-adrenoceptor stimulation on skeletal muscle vascular resistance and its relation to the level of background sympathetic activity. The isolated, separately perfused, neurally intact canine gracilis muscle preparation was used since it permits deliberate and quantifiable alterations in background sympathetic activity, as measured by skeletal muscle vascular resistance. Systemic intravenous UK-14304, a highly selective alpha 2-adrenoceptor agonist, produced a precipitous, neurogenic vasodilation that lowered vascular resistance below the subsequently denervated resistance, thus indicating that an active vasodilation was involved. The overall magnitude of the vasodilation was much greater in animals that had been hemorrhaged to elevate background sympathetic activity than in animals that had been transfused to lower background activity. The neurogenic vasodilation was unaffected by baroreceptor and cardiopulmonary receptor denervation and by prior cholinergic-receptor blockade of the gracilis muscle. Prior H1- and H2-histaminergic-receptor blockade, on the other hand, eliminated the active vasodilation but not a vasodilation down to the subsequently denervated resistance. Prior alpha 1-adrenoceptor blockade lowered resistance down to the subsequently denervated resistance and greatly attenuated the active vasodilation. The present study demonstrates that withdrawal of sympathetic activity by alpha 2-adrenoceptor stimulation produces an active vasodilation resulting from histamine release in skeletal muscle as well as a passive vasodilation resulting from lysis of peripheral vasoconstrictor tone.

Animals↗
PubMed1988

Cellular processing of pro-atrial natriuretic factor (pro-ANF): studies using an antiserum that selectively binds ANF-(99-126) after its cleavage from pro-ANF.

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a 15-17K prohormone, but circulates in plasma as a 3K, carboxy (C)-terminal fragment of the prohormone. The tissue location at which the cleavage of pro-ANF to its hormonal form occurs is unknown. In the present study, an immunological approach was taken to address this question. A polyclonal antiserum was generated which recognizes the hormonal form of ANF [ANF-(99-126)] only after its cleavage from the prohormone. This was accomplished by immunizing rabbits with a synthetic peptide corresponding to the seven amino (N)-terminal residues of ANF-(99-126) coupled to carrier protein via a C-terminal cysteine. This antiserum, anti-ANF-(99-105), demonstrated high affinity for ANF-(99-126) (IC50 = 170 pM), but displayed 100-fold less affinity for recombinant pro-ANF [ANF-(2-126)]. The N-terminal specificity of anti-ANF-(99-105) was evident by its failure to bind ANF-(103-126) at concentrations up to 100 nM. The specificity of anti-ANF-(99-105) for the hormonal form of ANF was examined by using thrombin to cleave pro-ANF and testing for the generation of anti-ANF-(99-105) immunoreactivity. Cleavage of atrial pro-ANF or 35S biosynthetically-labeled pro-ANF resulted in the production of immunoreactive material from the prohormone, whereas pro-ANF itself demonstrated no cross-reactivity with anti-ANF-(99-105). Anti-ANF-(99-105) could also recognize ANF released from the isolated perfused rat heart. When anti-ANF-(99-105) was used in immunohistochemical studies of rat atrial myocardium, no staining was observed in unfixed frozen sections. This suggests that proteolytic processing of pro-ANF is not an intracardiocytic event.

Amino Acid Sequence↗
PubMed1988

Electron microscopic localization of the beta-adrenergic receptor using a ferritin-alprenolol probe.

This report describes electron microscopic localization of the beta-adrenergic receptor using a beta-adrenergic receptor antagonist conjugated to ferritin. The conjugate was synthesized by reacting a carboxylic acid derivative of alprenolol with ferritin. The ferritin-alprenolol compound was shown to be effective in displacing specific [3H]dihydroalprenolol binding from rat erythrocyte membranes with a dissociation constant (Kd) of 25 nM. Rat erythrocyte ghosts were incubated with the compound and quantitative electron micrographic analysis yielded total binding of 1367 +/- 129 ferritin particles and nonspecific binding of 688 +/- 111 (six experiments). Thus, specific binding was 680 +/- 60 ferritin particles per red cell profile. Qualitative observations suggested that the particles were distributed randomly on the surface of the erythrocyte, although an occasional cluster was seen. A compound from another synthesis was shown be to effective in displacing specific [125I]iodocyanopindolol binding from neonatal rat cardiac myocyte membranes, with a dissociation constant of 13.8 nM, whereas native alprenolol had a dissociation constant of 1.3 nM. Neonatal rat cardiac myocytes were incubated with the compound and processed for electron microscopy. Total binding along the sarcolemmal membrane was 504 +/- 38 ferritin particles/100 micron of membrane and nonspecific binding was 301 +/- 26 ferritin particles/100 micron of membrane (seven experiments), yielding specific binding of 203 ferritin particles/100 micron of membrane. In additional studies, specific binding was inhibited 95% with 10(-5) M l-isoproterenol and 29% with d-isoproterenol, indicating stereoselectivity (seven experiments). The probe was distributed randomly along the sarcolemma with no preferential localization to coated pits or other membrane specializations. From measurements of the surface area of the average cardiac myocyte (732 micron 2), the specific binding of ferritin-alprenolol per 100 micron of membrane (203), and section thickness (0.08 micron), we calculated that cardiac myocytes had 18,575 beta-adrenergic membrane receptor sites. Thus, we have described a method for synthesizing and applying an electron-dense probe for electron microscopic localization of beta-adrenergic receptors. In these studies we determined the distribution of these receptors on rat erythrocyte ghosts and neonatal rat cardiac myocytes.

Alprenolol↗
PubMed1987

Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms.

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes.

Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiography. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease.

Animals↗
PubMed1987

Beta 2 microglobulin expression in keratoacanthomas and squamous cell carcinoma.

The cell surface expression of beta-2-microglobulin (beta 2 M) was investigated in 33 keratoacanthomas (KA) and 58 squamous cell carcinomas (SCC) to determine whether this antigen was expressed to a different extent in these two conditions and, thus, whether this constitutes a reliable and practical test for distinguishing them. Loss of beta 2 M expression was not a reliable feature for distinguishing between KA and SCC and seemed to be related more to the degree of cellular differentiation and maturation, than to malignancy as such.

Adult↗
PubMed1987

A serum protease cleaves proANF into a 14-kilodalton peptide and ANF.

Proatrial natriuretic factor (proANF), the 126-amino acid precursor of ANF, is the major storage form in mammalian atria. In contrast, two ANF peptides containing the 28- and 24-carboxyterminal residues of proANF have been isolated from rat plasma. Whether the cleavage of proANF in vivo to these ANF peptides occurs during or after its release into the circulation has not been determined. The latter possibility was suggested by our previous study where, by using a cultured rat cardiocyte preparation, we demonstrated that proANF is secreted intact into the culture medium. We now report that serum, but not plasma, contains a protease that specifically cleaves the 17-kdalton proANF to a 14-kdalton amino-terminal peptide and the carboxyterminal 3-kdalton circulating forms of ANF. The role of this proANF-cleaving enzyme in the generation of the biologically active ANF peptides remains to be defined. Its isolation and characterization should provide insights into its site of production and whether in vivo it is involved in the processing of circulating proANF.

Animals↗
PubMed1987

Age, race, diagnosis, and sodium effects on the pressor response to infused norepinephrine.

We studied the blood pressure responses to infused norepinephrine in 34 normotensive and 21 unmedicated subjects with essential hypertension. The two groups were similar in age, relative body weight, and urinary electrolyte excretion. Patients were studied on two extremes of dietary salt (200 mEq Na and 10 mEq Na per day). The dose-response curves were highly linear (p less than 0.00001) for both systolic and diastolic blood pressures. There was no evidence for an increased sensitivity to infused norepinephrine among the hypertensive subjects. On the other hand, older subjects had steeper slopes (p less than 0.005). Subjects on a high salt diet had steeper slopes than those on low salt diets (p less than 0.0025); this trend was especially apparent among blacks (p less than 0.005). Black and white hypertensive subjects responded to the high salt diet in opposite fashion: The blacks showed an increased pressor sensitivity (p less than 0.05), whereas the whites demonstrated a nonsignificant decreased pressor sensitivity. These results indicate that age, race, and salt effects must be meticulously controlled in studies of sympathetic nervous system physiology.

Adult↗
PubMed1987

Synthesis and characterization of arylamine derivatives of rauwolscine as molecular probes for alpha 2-adrenergic receptors.

The selective alpha 2-adrenergic receptor antagonist rauwolscine was structurally modified to yield a series of arylamine carboxamide derivatives, which were investigated as potential molecular probes for the localization and structural characterization of alpha 2-adrenergic receptors. The arylamine carboxamides differ in the number of carbon atoms separating the reactive phenyl moiety from the fused ring structure of the parent compound, rauwolscine carboxylate. Competitive inhibition studies with [3H]rauwolscine in rat kidney membranes indicate that the affinity for the carboxamide derivatives is inversely related to the length of the carbon spacer arm with rauwolscine 4-aminophenyl carboxamide (zero carbon spacer arm; rau-AMPC) exhibiting the highest affinity (Kd = 2.3 +/- 0.2 nM). Radioiodination of rau-AMPC yields a ligand, 125I-rau-AMPC, which binds to rat kidney alpha 2-adrenergic receptors with high affinity, as determined by both kinetic analysis (Kd = k2/k1 = 0.016 min-1/2.1 X 10(7) M-1 min-1 = 0.76 nM) and equilibrium binding studies (Kd = 0.78 +/- 0.16 nM). 125I-rau-AMPC was quantitatively converted to the photolabile arylazide derivative 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-azido-3-[125I]iodophenyl) carboxamide (125I-rau-AZPC). In a partially purified receptor preparation from porcine brain, this compound photolabels a major (Mr = 62,000) peptide. The labeling of this peptide is inhibited by adrenergic agonists and antagonists with a rank order of potency consistent with an alpha 2-adrenergic receptor binding site. Both 125I-rau-AMPC and the photolabile arylazide derivative, 125I-rau-AZPC, should prove useful as molecular probes for the structural and biochemical characterization of alpha 2-adrenergic receptors.

Adrenergic beta-Agonists↗
PubMed1987

Altered adrenergic control in hypercholesterolemia.

Vascular reactivity is altered by hypercholesterolemia and atherosclerosis. Animal studies have shown that serotonergic mechanisms of vasoconstriction are enhanced, while the changes with adrenergic mechanisms are still equivocal. Serum lipids are increased with beta-blocker and diuretic therapy for hypertension. Thus, the increase in serum lipids may be responsible for both structural and functional abnormalities of the vascular system in hypertensive patients. In contrast, recent reports have suggested that alpha 1-adrenergic receptor inhibition, useful in hypertension therapy, may exert a beneficial effect on plasma lipids. We have also collected preliminary data in our laboratory examining the effects of alpha 1-adrenergic receptor inhibition with prazosin on plasma cholesterol levels and vascular reactivity in monkeys with hypercholesterolemia. We observed large reductions in both total cholesterol and the very-low-density lipoprotein (VLDL) + low-density lipoprotein (LDL) fraction of cholesterol in these animals during treatment with prazosin. Thus, in addition to its sympatholytic action on the vasculature, alpha 1-adrenergic receptor inhibition appears to exert a beneficial effect in reducing total cholesterol levels.

Adrenergic alpha-Antagonists↗