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P Schnabel

Publications and source records attributed to P Schnabel.

At least 55 records · Page 3Linked to original sources
PubMed1996

Role of G-proteins in altered beta-adrenergic responsiveness in the failing and hypertrophied myocardium.

In the heart and other tissues, beta-adrenergic desensitization occurs during treatment with catecholamines. In heart failure, a strong sympathetic activation has been observed and is the cause of beta-adrenergic desensitization in this condition. On the receptor level, there is a downregulation of beta 1-adrenergic receptors as well as an uncoupling of beta 2-adrenoceptors. The latter mechanism has been related to an increased activity and gene expression of beta-ARK2 in failing myocardium leading to phosphorylation and uncoupling of receptors. In addition, an increase of inhibitory G-protein alpha-subunits (Gi alpha) has been suggested to be causally linked to adenylyl cyclase desensitization in heart failure. In contrast, the catalytic subunit of adenylyl cyclase, stimulatory G-protein alpha-subunits and beta gamma-subunits have been observed to be unchanged. Recently, evidence has been raised that increases of Gi alpha also depress adenylyl cyclase in compensated cardiac hypertrophy in monogenic and polygenic as well as in secondary hypertension. These increases of Gi alpha can suppress adenylyl cyclase in the absence of beta-adrenergic receptor downregulation. Since cardiac hypertrophy in pressure overload is a strong predictor of cardiac failure these observations indicate that adenylyl cyclase desensitization by Gi alpha could be a pathophysiologically relevant mechanism to contribute to the progression from compensated cardiac hypertrophy to heart failure.

Animals↗
PubMed1996

Heterotrimeric G proteins in heart disease.

Heterotrimeric G proteins couple many types of cell surface receptors to intracellular effectors such as enzymes or ion channels. In the mammalian heart, G protein-mediated signalling pathways are involved in the regulation of contractile force, heart rate, conduction velocity, and relaxation. In the first part of this review we summarize some important structural and functional features of receptors, G proteins, and effectors with special focus on the heart. In the second part, we review the current knowledge about alterations of G protein-mediated signalling in heart disease such as myocardial hypertrophy and heart failure.

Animals↗
PubMed1995

Mutational analysis of a putative polyphosphoinositide binding site in phospholipase C-beta 2.

The phosphatidylinositol 4,5-bisphosphate (PtdIns-P2)-regulated actin-binding protein gelsolin and most phosphoinositide-specific phospholipases C (PLCs) comprise a basic amino acid motif ((K/R)xxxKxK(K/R); x denotes any amino acid) which was previously suggested to represent a PtdInsP2-binding site commonly present in these proteins. We have challenged this hypothesis for PLC beta 2 by replacing one or several residues of this motif (KILIKNKK; residues 457-464) and examining the functional consequences of these alterations. The results show that the integrity of the basic motif is important for PtdInsP2 hydrolysis by PLC beta 2. Replacement of lysines 463 or 461 by arginine led to reduction or complete loss, respectively, of enzyme activity. The results provide further support to the concept that the function of the basic motif within the various PLCs is to bind the enzyme substrate PtdInsP2.

Amino Acid Sequence↗
PubMed1995

Characterization of putative polyphosphoinositide binding motifs from phospholipase C beta 2.

Several phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-regulated actin-binding proteins and most phosphoinositide-specific phospholipases C (PI-PLCs) comprise a basic amino acid motif (KxxxKxKK, where x denotes any amino acid), which was previously suggested to represent a PtdInsP2-binding site commonly present in these proteins. We have shown earlier that a peptide corresponding to amino acids 448-464 of human PLC beta 2 (LPSPEDLRGKILIKNKK, peptide P1) markedly and specifically stimulated the activity of this enzyme [Simões et al. (1993) FEBS Lett. 331, 248]. Here, we present a detailed analysis of the effects of various peptides related to peptide P1 aimed at understanding the mechanisms of peptide-mediated PLC beta 2 stimulation. Peptide KILIKNKK (P2), which comprises only the basic amino acid consensus motif, also stimulated PLC beta 2, although higher concentrations were required to observe this stimulatory effect. The effects of P1 and P2 were not additive, indicating that the two peptides affect PLC beta 2 activity via the same mechanism. Peptide LPSPEDLRG (P3), composed of the amino-terminal half of P1, did not affect the activity of PLC beta 2. Peptide KILIKNKKQFSGPTSS (P4), which includes the nine amino acids flanking the carboxy-terminus of the KILIKNKK motif within the sequence of PLC beta 2, stimulated the enzyme but was indistinguishable in potency from P2. Circular dichroism analysis revealed that peptide P1 changes its conformation in the presence of PtdInsP2 but not in the presence of other phospholipids including phosphatidylinositol 4-phosphate. The results suggest that the basic amino acid sequence physically interacts with PtdInsP2.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Mutations of signal-transducing G proteins in human disease.

Heterotrimeric guanine nucleotide binding proteins (G proteins) couple a large number of cell surface receptors to their intracellular effector molecules, such as enzymes or ion channels. Mutations of G proteins can lead to either activation or inactivation of the corresponding signal transduction pathway and thus cause clinical symptoms. Mutations of heterotrimeric G proteins have been found in a number of endocrine tumors, the McCune-Albright syndrome, Albright's hereditary osteodystrophy, and a combination of precocious puberty and pseudohypoparathyroidism Ia. The identification of the molecular defects underlying the above disorders and the investigation of their functional consequences for metabolism and growth regulation have been the subject of many studies over the past few years. A close understanding of these pathophysiologic mechanisms is crucial for the development of therapeutic strategies.

Amino Acid Sequence↗
PubMed1995

Cardiocirculatory effects of acutely increased intracranial pressure and subsequent brain death.

Hemodynamic instability and functional impairment of the donor heart are currently reported problems in organ transplantation. Actual shortage of potential donor hearts continues to raise controversial discussion about adequate donor management with regard to graft quality. In an experimental open chest model, physiopathologic effects of acutely induced, irreversible intracranial hypertension (AIIHT) were investigated in situ with respect to hemodynamics, cardiac pump and muscle function, and hormonal parameters. Acutely induced irreversible intracranial hypertension was induced by rapid inflation of a subdural balloon catheter in 10 anesthetized dogs, four animals serving as controls. The observation period in both groups was 300 min. Cardiocirculatory stability was maintained by continuous crystalloid volume substitution without the use of inotropic or pressor agents. After AIIHT, three characteristic hemodynamic response phases have been observed: 1) The "acute hyperdynamic phase" lasting up to 15 min with marked increases of heart rate (HR), left ventricular pressure (LVP), cardiac output (CO) and myocardial contractility indices, 2) At the end of the "early restabilization phase", (60 min), these parameters returned close to control levels, except HR (+50%) and systemic vascular resistance (SVR) (-40%), 3) During the "late restabilization phase", filling pressures, LVP and CO remained within control limits at low SVR, contractility indices showed a decreasing tendency. All assessed plasmatic hormones (Catecholamines, triiodothyronine (T3), thyroxine (T4), adrenocorticotropic hormone (ACTH), cortisol and anti-diuretic hormone (ADH) showed a continuous fall to levels significantly below control over the phases of restabilization. Acutely induced irreversible intracranial hypertension leads to multifactorial hemodynamic and hormonal changes. At low SVR, cardiac pump function was preserved exclusively by continuous volume substitution, while myocardial contractility indicated a slight decrease. From this observed hemodynamic and functional state within the donor organism, no reliable prediction on graft functional capacity can be made.

Animals↗
PubMed1994

Radioimmunochemical quantification of Gi alpha in right and left ventricles from patients with ischaemic and dilated cardiomyopathy and predominant left ventricular failure.

An increase of Gi alpha-related pertussis toxin substrates has been observed in the failing myocardium. In order to quantify the protein expression of Gi alpha directly, we developed a fast radioimmunoassay using the iodinated synthetic peptide 125I-KENLKDCGLF. beta-adrenoceptors were studied with 125I-cyanopindolol binding for comparison. Immunoblot experiments using recombinant G-protein alpha-subunits showed that DS4 immunostained the G-protein alpha-subunits with a rank order of potency rGi alpha 1 = rGi alpha 2 > rGo alpha >> rGi alpha 3. The G-protein alpha-subunits recognized by DS4 in human ventricular membranes comigrated with rGi alpha 1 and rGi alpha 2. The radioimmunoassay had a sensitivity of 2.5 micrograms/ml transducin alpha with an interassay variation of less than 10%. The non-labelled peptide selectively competed with the myocardial 40 kDa membrane protein for binding to the antiserum DS4. Radioimmunochemical quantification of Gi alpha from cardiac membranes showed that in left ventricular membranes (LV) from dilated cardiomyopathy (DCM), there was an increase of Gi alpha by 138.5% when related to mg protein and 135% when related to 3H-ouabain binding sites as membrane marker. In LV from ischaemic cardiomyopathy (ICM), the increase was smaller (58.4%) when related to mg protein compared to the increase of Gi alpha when related to 3H-ouabain binding sites as membrane marker (155% v NF). In contrast, in the right ventricles (RV) there was no increase of Gi alpha in ICM or DCM. The numbers of beta-adrenoceptors were reduced in RV and LV of both, ICM and DCM. It is concluded that the radioimmunoassay may become an important tool for studying the expression of Gi alpha-protein levels and changes thereof in pathological conditions. The amount of immunodetectable Gi alpha-proteins is increased in failing LV due to DCM and ICM but not in RV, while beta-adrenoceptor down-regulation occurred in RV and LV in both conditions. These findings might indicate that the liability of the LV but not of RV to express Gi alpha-proteins may be increased in predominant LV heart failure. Alternatively, the underlying mechanism, e.g. sympathetic activation, may be regulated locally in the failing heart producing different changes in adjacent chambers.

Adolescent↗
PubMed1993

Mutational analysis of phospholipase C-beta 2. Identification of regions required for membrane association and stimulation by guanine-nucleotide-binding protein beta gamma subunits.

Members of the beta isozyme subfamily of the phosphoinositide-specific phospholipases C (PLC beta) have recently been shown to be stimulated by both guanine-nucleotide-binding protein alpha and beta gamma subunits. The alpha subunits of the Gq class activate PLC beta isozymes in the order of PLC beta 1 > or = PLC beta 3 >> PLC beta 2, which is different from the order of PLC beta 3 > PLC beta 2 > PLC beta 1 for beta gamma subunit stimulation. The C-terminal region of PLC beta 1, in particular the sequence between Thr903 and Leu1142, has been shown to be involved in interacting with activated alpha q subunits and to contain a region required for efficient membrane association of PLC beta 1 [Park, D., Jhon, D.-Y., Lee, C.-W., Ryu, S. H. & Rhee, S. G. (1993) J. Biol. Chem. 268, 3710-3714, and Wu, D., Jiang, H., Katz, A. & Simon, M. I. (1993) J. Biol. Chem. 268, 3704-3709]. To examine the structure-function relationships of a PLC beta isozyme highly sensitive to beta gamma subunit stimulation, we have altered the cDNA of PLC beta 2 by site-directed mutagenesis and have examined the effects of these structural alterations on the functional properties of the mutant polypeptides. The results show that the C-terminal region of PLC beta 2 downstream of Phe818, which corresponds to Tyr816 of PLC beta 1, contains a region essential for membrane association, but is required neither for the interaction of PLC beta 2 with Ca2+ and the phospholipid substrate, nor for beta gamma subunit stimulation of PLC beta 2. These data suggest that PLC beta isozymes are activated by alpha q and beta gamma subunits via distinct domains.

Base Sequence↗
PubMed1993

A peptide corresponding to a potential polyphosphoinositide binding site of phospholipase C-beta 2 enhances its catalytic activity.

A peptide corresponding to a basic consensus amino acid motif present in both actin-binding proteins and phosphoinositide-specific phospholipases C was synthesized and its effect on the activity of a recombinant phospholipase C-beta 2 (PLC beta 2) expressed in baculovirus-infected insect cells was studied. The peptide markedly and specifically stimulated the activity of the enzyme. This stimulatory effect required a particular primary and/or secondary structure of the peptide and occurred without lowering the affinity of the enzyme for Ca2+. The function of the PLC beta 2 segment corresponding to the peptide might be to bind and offer the substrate to the catalytic domain of this enzyme in a more favorable configuration or, alternatively, to interact with a hypothetical inhibitory constraint.

Amino Acid Sequence↗
PubMed1992

Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits.

Hydrolysis by phospholipase C (PLC) of phosphatidylinositol 4,5-bisphosphate is a key mechanism by which many extracellular signalling molecules regulate functions of their target cells. At least eight distinct isozymes of PLC are recognized in mammalian cells. Receptor-controlled PLC is often regulated by G proteins, which can be modified by pertussis toxin in some cells but not in others. In the latter cells, PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, may be activated by members of the alpha q-subfamily of the G protein alpha-subunits. An unidentified PLC in soluble fractions of cultured human HL-60 granulocytes is specifically stimulated by G protein beta gamma subunits purified from retina and brain. Identification of a second PLC-beta complementary DNA (PLC-beta 2) in an HL-60 cell cDNA library prompted us to investigate the effect of purified G protein beta gamma subunits on the activities of PLC-beta 1 and PLC-beta 2 transiently expressed in cultured mammalian cells. We report here that PLC-beta 1 and PLC-beta 2 were stimulated by free beta gamma subunits and that PLC-beta 2 was the most sensitive to beta gamma stimulation. Thus stimulation of PLC by beta gamma subunits is isozyme-selective and PLC-beta 2 is a prime target of beta gamma stimulation. Activation of PLC-beta 2 by beta gamma subunits may be an important mechanism by which pertussis toxin-sensitive G proteins stimulate PLC.

Animals↗
PubMed1992

Stimulation of phospholipase C by a mutationally activated G protein alpha 16 subunit.

Alpha 16, a member of the alpha q subfamily of G protein alpha subunits, was recently identified in human hematopoietic cells. In order to elucidate the function of this novel alpha subunit, we cloned and mutagenized its cDNA to obtain a constitutively active protein. COS-1 cells were transfected with both wild-type and mutant cDNAs. Expression was confirmed by immunoblotting using a rabbit antiserum raised against the C-terminal decapeptide of alpha 16. The constitutively activated mutant alpha 16-R186C caused a two-fold increase in the formation of inositol trisphosphate in intact COS-1 cells, while the wild-type alpha 16 subunit had no effect. We conclude that alpha 16 is involved in coupling cell surface receptors of human hematopoietic cells to stimulation of phospholipase C.

Animals↗
PubMed1992

Down and out: social marginality and homelessness.

In the last ten years the problem of homelessness has been on the increase. Compared to the situation about sixty years ago the homeless of today congregate more in the centres of the big cities, adhere to a different lifestyle and are socially, culturally and ethnically more diverse. Some indications of the scale of the problem in the Netherlands are given, but the focus of the paper is on the role of psychiatry: on the one hand, in providing services to the mentally ill among the homeless and, on the other hand, in protecting communities against people who are considered bothersome or unfit for civil life. The development of systems of comprehensive care for different categories of socially marginal people is discussed.

Community Mental Health Services↗
PubMed1990

The puff-specific Drosophila protein Bj6, encoded by the gene no-on transient A, shows homology to RNA-binding proteins.

The DNA coding for the puff-specific protein Bj6 has been isolated by expression cloning. The gene is localized in 14C1,2 on the X chromosome and is expressed ubiquitously during embryonic development with prominent expression during the first 12 h of embryogenesis. cDNA and genomic clones have been sequenced and show a single open-reading frame of 2.1 kb length, coding for a Mr = 77,000 basic protein. In the aminoterminal half of the protein we detect stretches of repeated amino acids, centrally a region with homology to RNA-binding proteins containing the RNP 1 and RNP 2 consensus motif of RNA binding proteins, and the carboxyterminal part is rich in charged amino acids. The Bj6 protein is a product of the gene no-on transient A, a gene required for normal vision and courtship behaviour in Drosophila.

Amino Acid Sequence↗
PubMed1990

Improvement of cholera toxin-catalyzed ADP-ribosylation by endogenous ADP-ribosylation factor from bovine brain provides evidence for an unchanged amount of Gs alpha in failing human myocardium.

The aim of the present study was to investigate whether or not alterations of Gs alpha can be detected with cholera toxin-induced ADP-ribosylation in myocardial membranes from patients with heart failure. Therefore, Gs alpha was radiolabeled by cholera toxin-catalzyed (32P)ADP-ribosylation with (32P)NAD as substrate. In membranes from left ventricular myocardium of six patients with dilated cardiomyopathy classified as NYHA IV and three samples from two non-failing donor hearts, labeling was too weak to allow detection of possible changes in the amount of Gs alpha. Therefore, the cytosolic small molecular weight G protein ARF (ADP-ribosylation factor), a cofactor for cholera toxin-induced ADP-ribosylation of Gs alpha, was partially purified from bovine cerebral cortex. ARF activity was quantified by its ability to enhance auto-ADP-ribosylation of cholera toxin A1-subunit. Gs alpha was identified by comparing the ADP-ribosylation patterns of myocardial membranes, membranes prepared from human leukemia (HL 60) and S 49 mouse lymphoma wild type cells (45 kDa-band present) with membranes of the Gs alpha-deficient S 49 variant cyc- (45 kDa-band missing). In the presence of ARF, specific radiolabeling of the Mr 45,000 subtype of Gs alpha was markedly enhanced. The amounts of Gs alpha as measured by cholera toxin-dependent (32P)-ADP-ribosylation in the presence of ARR were similar in failing and nonfailing human hearts. It is concluded that factors other than Gs alpha are responsible for the altered regulation of the adenylate cyclase complex in heart failure. Moreover, by enhancing cholera toxin-catalyzed ADP-ribosylation, endogenous ADP-ribosylation factor from bovine brain appears to be a useful tool to study Gs alpha even in tissues in which the labeling of Gs alpha is rather weak.

ADP-Ribosylation Factors↗
PubMed1990

Increase of Gi alpha in human hearts with dilated but not ischemic cardiomyopathy.

In myocardial membranes from hearts with dilated cardiomyopathy (DCM), there was a 37% increase of the Gi alpha-protein as measured by 32P-ADP-ribosylation of a approximately 40 kDa pertussis toxin substrate. Immunoblotting techniques also showed increased amounts of Gi alpha in DCM. In hearts with ischemic cardiomyopathy (ICM), Gi alpha was not altered compared with nonfailing myocardium (NF). Basal and Gpp(NH)p-stimulated adenylate cyclase activity was reduced in DCM but not in ICM. The number of beta-adrenoceptors was similarly reduced both in DCM and ICM compared with NF. Alterations of m-cholinoceptors or A1-adenosine receptors did not occur. Consistently, "indirect" negative inotropic effects of the m-cholinoceptor agonist carbachol and the A1-adenosine receptor agonist R-PIA were not different in ICM, DCM, and nonfailing myocardium. In ICM and DCM, there was a marked reduction of the positive inotropic responses to isoprenaline and milrinone. However, there was a further reduction in DCM compared with ICM. It is concluded that the increase of Gi alpha is accompanied by a reduction of basal and guanine-nucleotide-stimulated adenylate cyclase activity. Alterations of m-cholinoceptors and A1-adenosine receptors do not appear to be involved. The further decrease of the positive inotropic effects of isoprenaline and milrinone in DCM provides evidence that the increase of Gi alpha is functionally relevant in DCM but not ICM and hence might contribute to the reduced effects of endogenous catecholamines and exogenous cAMP-dependent positive inotropic agents in the former but not the latter condition.

Adenosine Diphosphate Ribose↗