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H Porzig

Publications and source records attributed to H Porzig.

At least 37 records · Page 2Linked to original sources
PubMed1990

Regulation of beta-adrenergic responses during in vitro differentiation of mouse erythroleukemia cells.

Dimethyl sulfoxide (DMSO)-induced erythroid differentiation of Friend mouse erythroleukemia (MEL) cells is associated with a marked transient modulation of catecholamine sensitivity. Within 24 h after induction and well before the onset of hemoglobin synthesis, we observed a 3-fold increase in beta-receptor density and a more than 10-fold increase in receptor-coupled cAMP formation. During the following 4 days, in parallel with the development of normoblast-like cells, receptor numbers returned to preinduction levels while catecholamine-dependent cAMP formation remained significantly elevated. Simultaneously, the apparent potency of the beta-adrenoceptor agonist isoprenaline increased 10-fold. Improved receptor-cyclase coupling is probably due to a major shift in the expression of Gi and Gs regulatory proteins. Bacterial toxin-mediated ADP-ribosylation of membrane proteins suggests that the dominating species in native cells is Gi (Gsa:Gia = 1.7). By contrast, Gs predominates in differentiated cells (Gsa:Gia = 1.8:1). Receptor-independent forskolin-stimulated cAMP formation showed a pronounced, albeit transient, decrease during differentiation. We suggest that these changes in cellular cAMP responses may be important for transient positive or negative cooperative interactions between hormones and growth factors in the course of erythroid cell development.

Animals↗
PubMed1990

Differential expression by nerve growth factor of two types of Ca2+ channels in rat phaeochromocytoma cell lines.

1. Two clones of rat phaeochromocytoma PC12 cells have been used to study the expression of Ca2+ channels and their possible involvement in neuronal differentiation. One clone differentiated morphologically when exposed to nerve growth factor (NGF) for 4 days (PC12 cells), while the other clone was insensitive to NGF, but differentiated morphologically in the presence of ouabain (0.1 mM) for 7 days (PC12-mutant cells). 2. Whole-cell Ba2+ currents through Ca2+ channels were measured in PC12 cells at a test potential (Et) of +10 mV, from two holding potentials (Eh) of -90 and -30 mV (I-90 and I-30). NGF-induced differentiation increased I-90 by 248% and I-30 by 133%. The cells that differentiate in the presence of ouabain had only small, if any, Ba2+ currents that did not appear to change during morphological differentiation or after the addition of NGF. 3. Barium currents in PC12 cells could be separated into two components by selective antagonists. The component of I-90 that could be inhibited by omega-conotoxin GVIA (omega-CgTX) in NGF-differentiated cells was 458 +/- 84 pA (mean +/- S.E.M.), compared with 79 +/- 44 pA in native cells. I-30 was reduced by 50 +/- 17 pA in NGF-treated cells and was virtually insensitive to the toxin in native cells. By contrast, the dihydropyridine (DHP) isradipine reduced I-30 in NGF-treated cells by 30 +/- 8 pA and in native cells by 20 +/- 3 pA. 4. Radioligand binding studies with 125I-omega-CgTX in PC12 cell membrane fragments and in PC12 cells showed a 2- to 3-fold increase in maximal binding capacity after NGF exposure, while mutant cells showed no such change in binding capacity after treatment with NGF or ouabain. Staurosporine inhibited the effect of NGF on 125I-omega-CgTX binding. [3H](+)-isradipine binding capacity was increased 1.8-fold by NGF in depolarized PC12 cells while no change was observed in mutant cells after NGF or ouabain. There was no interaction between omega-CgTX and DHP binding sites. 5. Both the electrophysiological and the binding data indicate a preferential expression of omega-CgTX-sensitive Ca2+ channels (N type) over isradipine-sensitive channels (L type) in PC12 cells treated with NGF. By contrast, ouabain-induced differentiation of a mutant PC12 cell line, that lacks functional NFG receptors, was not associated with the expression of Ca2+ channels.

Adrenal Gland Neoplasms↗
PubMed1990

Regulation of beta-adrenergic responsiveness during erythroid differentiation of Friend erythroleukemia cells.

Mouse erythroblastoma cells were used as a model system to study sensitivity and regulation of the beta-adrenergic system during DMSO-induced differentiation from the proerythroblast to the normoblast stage. Differentiation was characterized by a initial marked increase in hormonal sensitivity lasting 24 to 40 h followed by a gradual loss of beta-adrenergic responsiveness. These changes are mainly due to a rapid but transient increase in receptor density (4 fold) and a marked shift of the membrane concentrations of the transmembrane signalling proteins Gs and Gi. The Gs to Gi ratio changed from 1:7 in native MEL cells to 2:1 in differentiated cells. By contrast, fusion experiments between rat reticulocytes and and MEL cells indicated that cytosolic factors, while not prominently involved in the regulation of the beta-adrenergic system of differentiating MEL cells, may be responsible for the rapid loss of cyclase activity during reticulocyte maturation, the last step in red cell formation.

Adrenergic beta-Antagonists↗
PubMed1989

Voltage-gated cardiac Ca channels as a target for new positive inotropic drugs.

The present state and future prospect of drug-induced activation of voltage-gated Ca channels as a means to improve cardiac contractility is shortly reviewed. All the presently available Ca channel activators are 1,4-dihydropyridine derivatives that bind with high affinity to both the open and the inactivated channel conformation. Nonselective simultaneous activation of cardiac and vascular smooth muscle L-type Ca channels prevents the use of these compounds as therapeutic agents. Possible strategies to overcome this dilemma include the use of suitable mixtures of a channel-activating drug with a channel-blocking drug and the exploitation of tissue-specific structural features of the channel for the development of new selective nondihydropyridine drugs. The development of functionally active, partially tissue-specific antibodies directed against Ca channel subunits suggests that in the near future the second approach may become feasible.

Animals↗
PubMed1988

mRNA-induced expression of the cardiac Na+-Ca2+ exchanger in Xenopus oocytes.

Xenopus oocytes were injected with total mRNA isolated from hearts of 1-day-old chicks. After 5 days of incubation the follicular cell layers were removed and the oocytes were loaded with Na+ by incubation in hypertonic EGTA solution at 37 degrees C. The Na+-loaded oocytes accumulated 45Ca2+ from a Na+-free medium at a 3-18-fold higher rate than noninjected oocytes or oocytes injected with control solution containing no mRNA. Oocytes not subjected to the Na+-loading procedure showed no mRNA-dependent 45Ca2+ uptake. Size fractionation of the mRNA using sucrose density gradient centrifugation under denaturing conditions led to the identification of a 25 S fraction competent for induction of the Na+-Ca2+ exchange system.

Animals↗
PubMed1988

Potential-dependent allosteric modulation of 1,4-dihydropyridine binding by d-(cis)-diltiazem and (+/-)-verapamil in living cardiac cells.

We have studied allosteric effects of the Ca channel blockers d-(cis)-diltiazem, (+/-)-verapamil, and (S)- and (R)-devapamil on the specific binding of the 1,4-dihydropyridine derivative (+)-[3H]PN 200-110 to intact tissue cultured rat heart cells. In polarized cells (membrane potential, -38 +/- 4 mV) d-(cis)-diltiazem (5 microM) increased the affinity of the radiolabel 2-4-fold causing 100-187% enhancement of binding at (+)-PN 200-110 concentrations below 0.5 nM. (+/-)-Verapamil (0.1-3 microM) had a similar, although smaller, effect on (+)-PN 200-110 binding. The two enantiomers of devapamil were without effect. In depolarized cells (membrane potential, 0 mV) d-(cis)-diltiazem had a small and the phenylalkylamines a strong inhibitory effect on (+)-PN 200-110 binding, mainly due to a reduction of binding affinity. At 50% receptor occupation by the radioligand, (R)-devapamil, (+/-)-verapamil, and (S)-devapamil displaced 40, 55, and 75%, respectively, of specifically bound radiolabel. Half-maximal effects were reached with 50, 20, and 4.5 nM, respectively, of the three compounds. Compared with nominally Ca-free medium (containing 3-5 microM Ca), addition of 1.25 mM CaCl2 caused an increase in the maximal binding capacity for (+)-PN 200-110 in both polarized and depolarized cells. However, Ca had only marginal effects on the allosteric interactions between (+)-PN 200-110, d-(cis)-diltiazem, and verapamil. We conclude from our results that positive cooperative interactions between Ca channel blockers prevail under conditions in which the voltage-dependent Ca channel can fluctuate between closed, open, and inactivated states. Negative cooperativity is usually observed under conditions in which all channels are inactivated (depolarized cells, fragmented membranes). Therefore, it is impossible to predict the type and the extent of allosteric interactions in vivo from studies in cell homogenates.

Allosteric Regulation↗
PubMed1987

Voltage-dependent cooperative interactions of calcium channel ligands in intact cardiac cells.

Voltage-dependent Ca channels were studied in living, tissue cultured rat heart cells using patch clamp analysis of single channels and radioligand binding studies at different membrane potentials. Ca channel activating and blocking dihydropyridines (DHP) show cooperative interaction and, therefore, bind to at least two different binding sites on the channel protein. Cooperative interactions between activating and blocking DHP, between DHP and verapamil and between DHP and diltiazem are all voltage dependent. The type of interaction in polarized cells and hence, the possible effects in vivo cannot be predicted from studies in cell homogenates.

Allosteric Site↗
PubMed1986

Studies on Ca channels in intact cardiac cells: voltage-dependent effects and cooperative interactions of dihydropyridine enantiomers.

We have investigated the effects of two oppositely acting enantiomers of the 1,4-dihydropyridine derivative 202-791 on voltage-dependent Ca channels by combining electrophysiological techniques and binding studies. The (S)-enantiomer of 202-791 promoting prolonged openings of single Ca channels, and thereby increasing transmembrane Ba currents, was classified as channel activator. The (R)-enantiomer favoring a closed state of the channel, and thereby reducing Ba currents, was classified as a channel blocker. Both compounds shifted the steady state current inactivation curve toward more negative potentials. At holding potentials positive to -20 mV, the Ca channel-activating effect of the (S)-enantiomer turned over into a blocking effect. In cells with normal resting potential the combination of the two enantiomers revealed a possible positive cooperative effect resulting in an enhancement of the open state probability of the channels. At depolarized holding potentials the activator enhanced the inhibitory effect of the blocker. Binding studies in intact cells were performed by using the radiolabeled channel-blocking dihydropyridine 3H-(+)-PN 200-110. The results showed a strong increase in binding affinity but no change in binding capacity when the cells were depolarized. Analysis of the interactions of (S)- and (R)-202-791 with this radioligand indicated stimulation of 3H-(+)-PN 200-110 binding by the (S)-enantiomer in polarized cells (membrane potential -38 +/- 4 mV). This effect could be attributed to an increase in binding affinity. The (R)-enantiomer had no such positive cooperative effect, but acted as a purely competitive ligand. Depolarization to 0 mV increased the apparent affinity of both enantiomers by factors of 38 (blocker) and 12 (activator), but abolished the cooperative effect of (S)-202-791 on the binding of the radioligand. Ca ions had little effect on the binding of 3H-(+)-PN 200-110 in polarized cells. However, in the presence of the activating (S)-enantiomer, Ca transformed the usual hyperbolic binding isotherm of the radioligand into a strongly sigmoid curve. Sigmoidicity was minimal with 3-5 microM Ca and maximal with 0.5 mM Ca. Together these data demonstrate homotropic and heterotropic cooperative interactions between channel activator and channel blocker. They indicate that at least two high affinity binding sites for dihydropyridines are associated with voltage-dependent Ca channels. Voltage dependence of both--binding affinity and cooperativity--suggests that these binding sites are located close to a structural component of the channel which is involved in the potential-sensitive gating process.

Aging↗
PubMed1986

The voltage-dependent effect of 1,4-dihydropyridine enantiomers on Ca channels in cardiac cells.

We studied voltage-dependent binding and action of 1,4-dihydropyridine enantiomers (Sandoz (+)-(S) & (-)-(R)-202-791) in intact cardiac cells. Drug action was studied by patch clamp method. (+)-enantiomer primarily prolonged open time of Ca channel, thus enhancing Ca currents, while (-)-enantiomer predominantly favored closed state of the channel, reducing the currents. Electrophysiological study also revealed that steady-state inactivation was greatly enhanced by both enantiomers. Therefore, it seems that both enantiomers have a capability of activating and blocking effects. Strongly voltage-dependent binding affinity of these enantiomers was revealed by displacement of 3H-(+)-PN200-110 with each enantiomers. Binding affinity of both enantiomers in depolarized cells was much higher than in polarized cells. These results indicate that both enantiomers have higher affinity when Ca channels are inactivated.

Animals↗
PubMed1985

Quantitative differences in the pharmacological effects of (+)- and (-)-cathinone.

The optically pure isomers of cathinone were prepared by separating synthetic cathinone racemate and used to study central and peripheral effects of these indirect sympathomimetics in rats and guinea pigs. The (-)-isomer was significantly more potent than the (+)-isomer in stimulating locomotor activity whereas no difference was observed with respect to their cardiac effects. In analogy to observations with (+)- and (-)-amphetamine such variable isomer discrimination may be due to different stereoselectivities of amine uptake mechanisms in the target tissues.

Alkaloids↗
PubMed1985

Regulation by 8-Br-cAMP of beta-adrenoceptors in cultured myocardial cells.

Primary cultures of myocardial cells from neonatal rats were exposed for up to 5 days to the cyclic AMP derivative 8-Br-cAMP. After one day of exposure to the nucleotide, an increase in specific binding capacity of the hydrophilic beta-adrenoceptor antagonist 3H-CGP 12177 was observed in intact cells. (-)-Isoprenaline displaced the radioligand from its binding site. Neither the KD of the antagonist, nor that of the agonist were significantly affected by 8-Br-cAMP. The loss of beta-adrenoceptors during desensitization by long term treatment of the cells with isoprenaline could be partially prevented by 8-Br-cAMP. Only in desensitized, but not in normal cells was isoprenaline-induced cAMP formation significantly enhanced by 8-Br-cAMP pretreatment. This may indicate that beta-adrenoceptors which appear during 8-Br-cAMP exposure are poorly coupled to the adenylate cyclase. Alternatively, the change in receptor density may be accompanied by alterations of other components in the beta-adrenergic system, e.g. an inhibition of the adenylate cyclase. We suggest that cAMP-dependent feed-back regulation of the beta-adrenergic system may play a role during postnatal myocardial differentiation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Plasma catecholamines, beta-adrenergic receptors, and isoproterenol sensitivity in endurance trained and non-endurance trained volunteers.

Six male non-endurance trained subjects (S) and six marathon runners (M) underwent graded treadmill exercise (T) and isoproterenol stimulation (I; 2 and 4 microgram X min-1). beta-adrenergic receptor density was additionally determined as the amount of 3H-Dihydroalprenolol (DHA) specifically bound on intact polymorphonuclear leucocytes. Heart rate, VO2 uptake, lactate, plasma noradrenaline, and adrenaline were estimated during T. Heart rate, stroke volume, cardiac output, as well as lactate, glucose, free fatty acids (FFA), and glycerol levels in the blood were determined during I. M showed the known training-dependent responses during T, such as lower heart rates, lactate levels, and plasma catecholamines at identical work loads, as well as higher VO2 max than S. I-induced cardiac output increase was quite similar in both groups. Stroke volume, however, increased significantly in M and stayed constant in S. Lactate decreased (S), glucose increased significantly (M), glycerol increased similarly in both groups, FFA rise was less marked in S. I-induced stroke volume response (I) may be indicative of a more economic regulation of heart work in M than S. Lactate decrease and less marked FFA increase, as observed in S, may be the result of a somewhat higher cardiac energy demand, dependent on less economic heart work. Higher DHA-binding as observed in M, as well as stroke volume response and glucose increase, may be indicators of a training-dependent rise in sensitivity to catecholamines. The unsolved question is, however, to what extent beta-receptor responses in intact blood cells are significant for receptor behavior in other organs.

Adult↗
PubMed1984

Recovery of beta-adrenoceptors and cyclic AMP response after long term treatment of intact heart cells with beta-blockers.

We have studied the recovery of receptor binding and of isoprenaline-stimulated cyclic AMP responses after chronic (2-5 days) exposure of tissue-cultured living rat heart cells to several beta-adrenoceptor antagonists. Most experiments were performed with [3H]- (+/-)-carazolol and [3H]-(+/-)-CGP 12177, as prototypes of high affinity lipophilic and hydrophilic ligands respectively. Chronic antagonist treatment did not alter the total number of receptors nor did it cause intracellular accumulation of the ligands. At the end of the treatment, radiolabelled antagonists were displaced either by 'infinite' dilution of the incubation medium or by competitive displacement with the non-labelled ligand (-)-timolol. In dilution assays dissociation of carazolol from specific sites was biphasic with t 1/2 values of 41 +/- 14 and 219 +/- 15 min. Dissociation of CGP 12177 was monophasic with t 1/2 of 102 +/- 2 min. Timolol enhanced the dissociation rates of both radioligands and suppressed the slow phase of carazolol dissociation. Isoprenaline-stimulated cyclic AMP formation did not recover in parallel with the release of the two antagonists from receptor binding sites. To reach about 80% of control values for receptor availability or cyclic AMP response required 3 h and 24 h washout periods, respectively, after carazolol (0.2 nM) treatment, or 1.5 and 12 h washout periods after CGP 12177 (4 nM) treatment. Such a 'decoupling' effect was not observed during recovery from chronic exposure to the antagonists, timolol and propranolol. We conclude that some antagonists cause a novel form of desensitization that is not linked to their partial agonistic potency. Moreover, carazolol-type drugs seem to induce an additional isomeric form of the beta-receptor that is not recognized by other antagonists. These observations could explain the well known discrepancy between long duration of action and rapid removal from the circulation of several antagonists in current therapeutic use.

Adrenergic beta-Antagonists↗
PubMed1984

Changes in beta-adrenoceptor binding properties and receptor-cyclase coupling during in vitro maturation of rat reticulocytes.

The loss of beta-adrenergic responsiveness during reticulocyte maturation was studied under tissue culture conditions in a defined cell population from rats. Initial beta-receptor density and receptor-mediated cAMP formation in culture medium (RPMI 1640) exceeded the values obtained in isotonic KC1-buffer by 56 and 120% respectively. During cell cultivation receptor density and hormonal responsiveness decreased rapidly by 40-50% of their initial values within the first 20 h. In the following 3 days the rate of loss varied between 10 and 15%/d. The same time course was observed for the maturation-dependent decrease in forskolin-stimulated cAMP formation. ATP depletion of cultivated cells caused a complete and irreversible loss of cAMP response within 90 min. Our results indicate that cell metabolism regulates the strength of the hormonal response. A defect in adenylate cyclase or in cyclase N-protein interaction seems to be rate-limiting for the functional inactivation of the beta-adrenergic system during reticulocyte maturation.

Adenosine Triphosphate↗