Search PubMed⌕ Search

Biomedical subjects

H Glossmann

Publications and source records attributed to H Glossmann.

At least 163 records · Page 9Linked to original sources
PubMed1979

Investigations on the mechanism of cyclic guanosine monophosphate increase due to depolarizing agents as studied with sea anemone toxin II in mouse cerebellar slices.

Sea anemone toxin II (ATX II) and MCD-peptide, like other depolarizing agents, raise the content of cGMP and to a lesser extent of cAMP in mouse cerebellar slices. Na+ influx and Ca2+ movement are involved in their mode of action, as indicated by the following observations: 1. The rise of cGMP due to ATX II, MCD-peptide and high potassium was diminished when Na+ had been replaced by Li+. 2. The effects of both toxins and veratridine, but not of high potassium stimulation were prevented by tetrodotoxin (TTX). 3. The cGMP accumulation due to both toxins was abolished in the absence of extracellular Ca2+. 4. The so-called Ca2+-antagonist (-)-D-600 blocked the increase of cGMP due to ATX II, MCD-peptide, veratridine and high potassium. 5. ATX II stimulated the 45Ca2+ uptake in mouse cerebellar slices which was prevented by TTX and (-)-D-600.

Adenosine Deaminase↗
PubMed1979

Alpha adrenoceptors in rat brain: direct identification with prazosin.

Tritiated prazosin was used to characterize high affinity binding sites with characteristics similar to alpha 1 adrenoceptors in rat brain membranes. These sites were compared with alpha 2 adrenoceptors labeled with tritiated clonidine. The prazosin sites had an association constant of 2 nM-1 and bound to ligand optimal around pH 7.0. The density of the sites was 300 fmoles per mg of protein; the half time of dissociation of prazosin was 7 min at 30 degrees C. The order or potencies of agonists, determined from binding-inhibition experiments with labeled prazosin, was: naphazoline greater than clonidine greater than adrenaline greater than noradrenaline greater than phenylephrine greater than alpha-methylnoradrenaline greater than dopamine. The order of potencies of antagonists was: prazosin greater than phenoxybenzamine greater than phentolamine greater than clozapine greater than yohimbine. Sodium ions and divalent cations as well as guanyl nucleotides have little or no effect on the binding of the labeled antagonist. This is in contrast to the binding of the labeled agonist clonidine (Glossmann and Presek, 1979a, 1979b). Labeled prazosin may be a useful tool to characterize alpha 1 adrenoceptors.

Animals↗
PubMed1978

Solubilization of a mammalian beta-adrenergic receptor.

Binding sites for iodohydroxybenzylpindolol with characteristics of a beta2-adrenergic receptor have been identified in a crude membrane fraction from guinea-pig lung. The binding sites could be solubilized by treatment of the membrane fraction with digitonin. Upon solubilization receptors retain their beta2-adrenergic type as indicated by the rank order of potencies of agonists in binding-inhibition experiments. The solubilized receptors demonstrate a marked increase in affinity for agonists compared with particulate receptors whereas antagonist affinity remains unchanged. Solubilized receptors are insensitive to divalent cations (Mg2+, Mn2+, Ca 2+) which increase the potency of agonists for particulate receptors. The effects of Mg2+ can be reversed by Gpp(NH)p in particulate preparations; Gpp(NH)p is ineffective for the solubilized preparation. These experiments establish that beta-adrenergic receptors can be solubilized even from crude mammalian membrane preparations. They also show that the mammalian beta-adrenergic receptor in situ is under constraints with respect to agonist affinity and is modulated by divalent cations and guanyl nucleotides in the intact membrane.

Adenylyl Cyclases↗
PubMed1977

Adrenal cortex adenylate cyclase. In vitro modification of the enzyme by cholera toxin.

Pretreatment of rat adrenal particulate fractions with cholera toxin in vitro markedly changed the properties of the membrane-bound adenylate cyclase. The basal activity of the enzyme was increased after cholera toxin treatment. The main action of the toxin was on the Vmax of the enzyme. In the absence of added GTP Lineweaver-Burk plots indicate a deviation from normal Michaelis-Menten kinetics with respect to substrate, the slopes being concave downward for control and toxin-treated membranes. Although hormonal stimulation of the enzyme was diminished in toxin-treated membranes, the hormone receptors were still functionally active as revealed after addition of Gpp(NH)p, GTP or GTPgammaS. The response to NaF was decreased in the toxin-treated membranes. Whereas GTP behaves as an antagonist (or a partial agonist with low intrinsic activity) compared to Gpp(NH)p in control membranes, it has similar intrinsic activity as Gpp(NH)p in the toxin-treated membranes. The results indicate that cholera toxin modification of the adenylate cyclase complex is located at the guanyl nucleotide sites or factors controlling the turnover of GTP at these sites. Cholera toxin modification may be a useful tool to investigate the role of guanyl nucleotide sites in the regulation of adenylate cyclase activity.

Adenosine Triphosphate↗
PubMed1977

Solid phase radioimmunoassay for cyclic AMP using staphylococcal protein A-antibody adsorbent.

A radioimmunoassay for cyclic AMP has been developed using protein A containing staphylococci as an immunoabsorbent. Protein A containing heat-killed staphylococci (Cowan I) are coated with rabbit antiserum raised against the 2'-O-succinyl derivative of cyclic AMP coupled to human serum albumin. After washing with a Tween 20 containing buffer, antibody coated staphylococci are diluted with heat-killed staphylococci devoid of protein A (staphylococcus epidermidis) and mixed with [125I]-2'-O-succinyl cyclic AMP tyrosine methyl ester, standards or unknowns. At the end of the incubation, separation of bound and free labelled antigen is achieved by bound and free labelled antigen is achieved by centrifugation. The results are comparable to those obtained with a precipitation assay using polyethylenglycol 6000. Acetylation prior to radioimmunoassay increases sensitivity about 80-fold. 50% depression of zero dose binding occurs at 15--16 femtomoles acetylated cyclic AMP. The crossreactivity with cyclic GMP, ATP, ADP, 5'-AMP and adenosine is extremely low. The present technique is an attractive alternative to the second antibody method or polyethylenglycol precipitation.

Acetylation↗
PubMed1976

Adrenal cortex adenylate cyclase. Is Ca2+ involved in ACTH stimulation?

The chelator EGTA inhibits the activation of bovine adrenal cortex adenylate cyclase by ACTH. Hormonal response is restored by addition of Ca2+, Mn2+ and other cations which are able to significantly reduce the concentration of uncomplexed EGTA in the adenylate cyclase assy medium. Time course studies reveal that the enzyme in the activated state (induced by adenylate cyclase reagents and hormone or by pretreatment with hormone) is resistant to EGTA inhibition.

Adenylyl Cyclase Inhibitors↗
PubMed1976

Beta-adrenergic receptors in guinea-pig myocardial tissue.

Stereospecific binding sites for (-) [3H]-alprenolol, a beta-adrenergic antagonist, have been identified in guinea-pig myocardial broken cell preparations. The concentration of the sites was 0.3 pmoles per mg of protein and the dissociation constant (at 37 degrees C) 10(-8) M. A close correlation between the ability of various beta-adrenergic antagonists to compete with tracer alprenolol binding and to block the response of isoprenaline-stimulated myocardial adenylate cyclase has been found. Low affinity sites for the labelled beta-adrenergic antagonist in contrast to stereospecific sites are heat stable and do not discriminate between the (-) and the (+) forms of the beta-adrenergic antagonists. Adenylate cyclase in guinea-pig myocardial tissue is poorly stimulated by isoprenaline or 5'-guanylylimidodiphosphate. This is attributed to a high basal activity which could be lowered by a preincubation at 37 degrees C.

Adenylyl Cyclases↗
PubMed1976

Adrenal cortex adenylate cyclase. In vitro acitivity of ACTH fragments and analogues.

The ability of ACTH fragments and of an ACTH analogue [9-tryptophan(o-nitrophenylsulfenyl)] corticotropin-(1-24)-tetracosapeptide[Trp-(Nps)9 ACTH1-24] to stimulate adenylate cyclase in bovine adrenal cortex membranes and a crude membrane fraction from rat adrenals has been determined. Partial agonists like Trp (Nps)9 ACTH1-24 displayed intrinsic activity in the rat adrenal preparation only if tested in the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p]. On the other hand, no addition of Gpp(NH)p was necessary to demonstrate intrinsic activity of Trp(Nps)9 ACTH1-24 for bovine adrenal cortex adenylate cyclase. A large decrease (15-fold) of the apparent Km values for ACTH1-24, ACTH1-23 and ACTH1-17 was observed with the rat adrenal preparation when Gpp(NH)p was added. The shift in apparent Km values for ACTH1-24 and ACTH1-23 for the bovine adrenal cortex adenylate cyclase system was small or insignificant when Gpp(NH)p was added. The observations suggest that the hormone receptor facilitates the action of guanylnucleotide sites in the membrane. When guanylnucleotide sites are occupied by Gpp(NH)p even weak interactions of the hormone receptor with e.g. partial agonists are propagated to the catalytic subunits of the adenylate cyclase complex resulting in enhanced activity. The differences in adenylate cyclase activation with hormone fragments or analogues and different target tissues may rather reflect the state of the coupling process involving guanylnucleotide binding sites of the isolated membrane fraction than differences in the receptor itself.

Adenylyl Cyclases↗
PubMed1975

Bovine adrenal cortex adenylate cyclase: properties of the particulate enzyme and effects of guanyl nucleotides.

The preparation of a partially purified plasma membrane fraction from bovine adrenal cortex is described. Adenylate cyclase in this particulate preparation retained high sensitivity to ACTH and is also stimulated by 5'-guanylyl-imidodiphosphate [Gpp(NH)p]. GTP, in contrast to Gpp(NH)p, had very little intrinsic activity to stimulate activity to stimulate adenylate cyclase. GTP could however, with high affinity, inhibit the Gpp(NH)p effects on adenylate cyclase. When the concentration of creatine phosphate, a component of the ATP-regenerating system in the adenylate cyclase assay mixture, was lowered from 20 to 2 mM (at 0.1 mM ATP, 5 MM MG2+) GTP, dGTP and other nucleotides like ITP and much less UTP or CTP gained considerable intrinsic activity in the presence of ACTH to stimulate adenylate cyclase. The apparent affinities of the nucleotides for ACTH-stimulated adenylate cyclase from bovine adrenal cortex (at 2 mM creatine phosphate) were, GTP = dGTP greater than Gpp(NH)p greater than Gpp(CH2)p (5'-guanylyl-beta, gamma-methylene-diphosphonate) greater than ITP greater than UTP greater than CTP. These findings indicate that regulatory nucleotide binding sites exist for bovine adrenal cortex adenylate cyclase. Their specificity is similar to the nucleotide sites modulating angiotensin binding in bovine adrenal cortex plasma membranes (Glossmann et al., 1974a). The regulatory nucleotide binding sites for the adrenal cortex adenylate cyclase complex can also be identified under conditions where only Gpp(NH)p has high intrinsic activity (e.g. at 20 mM creatine phosphate) but other nucleotides like GTP act as antagonists. Both stimulants, ACTH and Gpp(NH)p, appear to remain firmly bound to the particulate membrane preparation, as suggested by preincubation experiments.

Adenosine Triphosphate↗
PubMed1975

Adrenal cortex adenylate cyclase. Specific binding sites for 5'-guanylyl-imidodiphosphate in partially purified plasma membranes from bovine adrenal cortex.

Specific binding sites for 5'-guanylyl-imidodiphosphate [Gpp(NG)p] have been identified in a partially purified plasma membrane fraction from bovine adrenal cortex. The apparent affinity of Gpp(nh)p at 30 degrees C was 12muM-1 and the concentration of binding sites was 100 pmoles per mg of protein. Binding of Gpp(NH)p is inhibited by Mn2+ greater than Mg2+ greater than Ca2+ and enhanced by low concentrations of the chelators ethylenediamino-tetraacetic acid (EDTA) and ethylene glycolbis-(beta-aminoethylether))-N,N'-tetraacetic acid (EGTA). High concentrations of EDTA are inhibitory and at 2.5 mM EDTA binding of Gpp(NH)p is only 10% of that observed in the absence of the chelator. The bound labeled GTP analogue exchanged only slowly with the unlabeled nucleotide after a steady state has been reached. EDTA also releases the bound labeled Gpp(NH)p from its binding sites. The slow dissociation of Gpp(NH)p can explain the persistent activation of adenylate cyclase observed after pretreatment of bovine adrenal cortex plasma membranes with Gpp(NH)p and subsequent washing. It is suggested that at least parts of these binding sites are identical to the sites identified earlier as regulatory sites for angiotensin high-affinity receptors (Glossmann et. al., 1974a) and for ACTH-stimulated cyclase Glossmann and Gips, 1974).

Adenylyl Cyclases↗
PubMed1974

Cation dependence of high-affinity angiotensin II binding to adrenal cortex receptors.

The specific binding of monoiodinated angiotensin II by particulate receptors from the bovine adrenal cortex is enhanced by addition of sodium and potassium ions, but not other cations. In the presence of 140 millimolar sodium, increased uptake of angiotensin II by adrenal receptors is associated with the appearance of high-affinity binding sites with an association constant of 2 x 10(9) liters per mole.

Adrenal Glands↗