Yael Eilam

Expression of Opioid Receptors During Heart Ontogeny in Normotensive and Hypertensive Rats

BACKGROUND The opioidergic systems are involved in modulating nociceptive stimuli. In addition, the recent results suggest that endogenous and exogenous opioids could play a role in the modulation of blood pressure and cardiac functions. However, little is known regarding the expression and role of opioid-binding sites in the heart. The decreased sensitivity to noxious stimuli in hypertensive rats raises the possibility of different developmental pattern expression of opioid-binding sites in normotensive versus hypertensive rats. METHODS AND RESULTS Opioid receptor expression in hearts from hypertensive and normotensive rats was studied during heart development by binding assays. From P1 until P90, the development of the heart in the two rat strains was accompanied by a gradual increase in the density of kappa-opioid receptors. Hearts from hypertensive rats expressed significantly higher levels of kappa receptors compared with those of normotensive rats. At ages older than P7, mu-opioid receptors could not be detected in hearts of both strains, whereas delta-opioid-binding sites gradually increased until reaching adult levels. Seven-day-old cardiomyocyte cultures of both rat strains expressed similar densities of delta or kappa receptors to those observed in hearts from 7-day-old neonates. The mu-binding sites were not detected in cardiomyocytes cultures. Similar to the in vivo state, cultured myocytes from hypertensive rats had significantly higher levels of kappa-binding sites (1.5 fold) compared with those of normotensive rats. The kappa sites are pertussis toxin sensitive, and the state of coupling of the receptor to G protein is similar for the two rat strains. CONCLUSION The role of opioid-binding sites in the heart is not completely clear. Hypertensive rats are known to be less sensitive to noxious stimuli compared with normotensive rats. It is controversial whether the site if application of noxious stimuli plays an important role in the sensitivity to pain in hypertensive rats. We suggest that the opioidergic system could play a role in the modulation of blood pressure in addition to its known effect on nociception.

Inotropic action of σ receptor ligands in isolated cardiac myocytes from adult rats

High affinity binding sites for sigma receptor ligands were found in membranes of cardiac myocytes from adult rats. The sigma receptor ligand (+)-3-hydroxyphenyl-N-(1-propyl)piperidine ((+)-3-PPP) binds with a Kd of 17.9 +/- 4.0 nM and a Bmax of 275 +/- 32.1 fmol/mg protein. Competition experiments of (+)-pentazocine with [3H]1,3-di-O-tolylguanidine ([3H]DTG) binding yielded a Ki of 6.1 +/- 1.3 nM. The majority of the sites (> 80%) were of the sigma 1 subtype. Exposure of isolated cardiomyocytes from adult rats to (+)-3-PPP (10 nM-1.0 microM) caused a marked concentration-dependent increase in the amplitude of systolic cell contraction, reaching 149% of control level, with an apparent ED50 value of 4.5 nM. The increase in the contraction amplitude was markedly inhibited by pretreatment with verapamil or thapsigargin. An increase in the amplitude of [Ca2+]i transients, similar to that in the amplitude of cell contraction, was observed in indo-1-loaded cardiomyocytes exposed to 0.1 microM (+)-3-PPP. Exposure to 10 nM of haloperidol or (+)-pentazocine induced an increase in the amplitude of contraction, reaching 188% and 138% (respectively) of control level. A lower concentration of haloperidol or (+)-pentazocine (1 nM) did not induce an increase in the contraction amplitude but rather reduced the amplitude to 70-80% of control.

Highly selective σ receptor ligands elevate inositol 1,4,5-trisphosphate production in rat cardiac myocytes

Exposure of cardiac myocytes from adult rat ventricles to the highly selective, high affinity sigma receptor ligands BD-737 (0.1-100 nM) and BD-1047 (0.01-10 nM) caused potentiation of electrically-evoked amplitudes of contraction and Ca2q transients, while exposure to 100 nM BD-1047 caused attenuation of these amplitudes. In addition, BD-737 (1-100 nM) and BD-1047 (10-100 nM) caused an increase in the incidence of spontaneous twitches. These effects were inhibited when the incubation with BD-737 was done in the presence of the phospholipase C inhibitor, neomycin, or after pre-incubation with thapsigargin or caffeine which deplete the sarcoplasmic reticulum Ca2q stores. Inositol 1,4,5-trisphosphate (IP3) production in cardiac myocytes was determined by the IP3 binding protein assay. Both substances caused an increase in the intracellular concentration of IP3. BD-737 caused a rapid transient increase to 3.2-fold in 1 min and stabilization at 2.1-fold of control thereafter. BD-1047 caused a gradual increase reaching 4.4-fold after 5 min. The results suggest that the effects of these sigma receptor ligands on contractility and spontaneous contractions are mediated by activation of phospholipase C and elevation of intracellular IP3 level.

Leishmania major: Excreted factor, calcium ions, and the survival of amastigotes

Mouse macrophages infected with amastigotes of Leishmania major contain about 40% more intracellular exchangeable calcium than control macrophages. Similar elevation of intracellular exchangeable calcium was observed in macrophages engulfing red blood cells coated with purified excreted factor from L. major. The rate of cytolysis of red blood cells coated with excreted factor was significantly lower than that of uncoated controls. Excreted factor strongly binds calcium; thus, the possible role of a microenvironment rich in calcium bound to excreted factor within the phagolysosome in protecting the amastigotes may be considered.

Slowing down aging of cultured embryonal chick chondrocytes by maintenance under lowered oxygen tension.

Cultured epiphyseal-chondrocytes from embryonic chick may serve as a useful in vitro model to study aging processes in cartilage. The accelerated aging process in cultured chondrocytes is completed within a month and is manifested by typical changes in both cellular and extracellular compartments. Under common maintenance conditions, cells show a gradual loss of replicative capacity, increase in the rate of proteoglycan synthesis and age-dependent changes in the structure and composition of proteoglycan. An environmental factor--reduced oxygen tension--was found to slow down aging processes and preserve the young features of chondrocytes for a longer duration in culture. Cultures maintained under lower oxygen tension had higher proliferation rate, smaller cell size, lower rate of proteoglycan synthesis, and lower content of keratan sulfate side chains in the proteoglycan. In addition higher concentrations of free cytosolic calcium [Ca2+]in as compared to control cultures, was found. It is suggested that the increased proliferation rate and the decrease in proteoglycan synthesis caused by low oxygen tension may be signalled by the higher [Ca2+]in in these cells.

C-type natriuretic peptide has a negative inotropic effect on cardiac myocytes

C-type natriuretic peptide (CNP) has vasodilatory and antimitogenic actions, but its role in the control of cardiac function is unclear. We studied the effect of CNP on cultured, beating neonatal rat cardiac myocytes. CNP caused a significant reduction in the amplitude of contraction and a significant accumulation of intracellular cyclic GMP. The effect of a membrane permeable cyclic GMP on cell contraction was similar to that of CNP. CNP caused no change in Ca2+ transients. Blockade of natriuretic peptide receptors abolished the effects of CNP on contraction and accumulation of intracellular cyclic GMP. Blockade of cyclic GMP-dependent protein kinase abolished the effect of CNP on myocyte contraction. We conclude that CNP has a negative inotropic effect on neonatal rat cardiac myocytes. The effect of CNP is mediated via natriuretic peptide receptor(s) causing elevation of intracellular cyclic GMP which possibly activates protein kinase and causes attenuation of myofilament sensitivity to Ca2+.

Membrane effects of phenothiazines in yeasts. I. Stimulation of calcium and potassium fluxes

Application of trifluoperazine (10-50 microM) to suspensions of the yeast Saccharomyces cerevisiae induces the following effects. (1) A marked increase in the initial rate of 45Ca2+ influx into the cells, accompanied by an increase in the cellular content of calcium. This stimulation in 45Ca2+ influx (10-20-fold) is observed only in the presence of a metabolic substrate and is completely inhibited by LaCl3. The dose-response curves of the cellular accumulation of 45Ca2+ are of a bell shape, indicating a biphasic response. The concentration of the drug yielding maximal accumulation depends on the density of the cells in the suspensions. The results indicate that the stimulation of 45Ca2+ influx is mediated by an energy-dependent carrier-mediated process and not by the increase in the passive membrane permeability to Ca2+. (2) Efflux of K+ from the cells is induced. Removal of metabolic substrate abolishes the effect at concentrations of up to 35 microM and reduces it at higher concentrations. Addition of high concentrations of cations (K+, Na+, Mg2+) to the medium abolishes the stimulation of both K+ efflux and Ca2+ influx. Chloropromazine, thioridazine and chlorprothixene display similar effects, but at higher concentrations. The results are discussed in terms of two possible alternative mechanisms; (1) calmodulin-independent effects of trifluoperazine on cell membranes, or (2) inhibition of some calmodulin-dependent processes by low concentrations of trifluoperazine.

Decrease in cytosolic free Ca2+ and enhanced proteoglycan synthesis induced by cartilage derived growth factors in cultured chondrocytes

Cartilage-derived growth factors, enhance proteoglycan synthesis in cultured chick-embryo chondrocytes, and have almost no effect on cell proliferation. Addition of cartilage derived growth factors to cartilage cells loaded with the fluorescent Ca2+ indicator quin 2, caused a rapid, concentration dependent decrease in cytoplasmic free Ca2+. This decrease persisted also in Ca2+-free medium, indicating that it is not mediated by a decrease in the passive permeability of cell membrane to Ca2+. Addition of the Ca2+ ionophore A23187, with or without cartilage derived factors, caused an increase in cytoplasmic free Ca2+ together with inhibition of proteoglycan synthesis and enhanced cell proliferation. The results may indicate that whereas cell proliferation in chondrocytes is signaled by an increase in cytoplasmic Ca2+ ([Ca2+]in), proteoglycan synthesis is signaled by a decrease in [Ca2+]in. The data lead to suggesting a mechanism for antagonistic regulation of cell proliferation and the expression of the differentiated state.

A simple resolution of the kinetic anomaly in the exchange of different sugars across the membrane of the human red blood cell.

Abstract 1. 1. Using human red blood cells, we measured the time courses of efflux of labelled mannose, glucose or galactose (all at 130 mM) into equimolar concentrations of different sugars. 2. 2. We confirmed that the rates of exchange of mannose with mannose and galactose with galactose are somewhat slower than that of glucose with glucose at the particular concentration studied. The rates of exchange for glucose into mannose and for glucose into galactose are (we confirm) faster, but those for mannose into glucose and galactose into glucose are (we show) slower than for the exchange of either glucose into glucose, mannose into mannose or galactose into galactose. 3. 3. We determined directly, using the single sugars alone, the kinetic parameters K m and V for exchange transport. The different sugars demonstrated different values for V as well as for K m . 4. 4. Based on the values obtained, the theoretical time course of the exchange between pairs of sugars at 130 mM was computed according to the conventional carrier model and the recently proposed tetramer model. The experimental results fitted well with the predictions of the tetramer model and to a lesser extent with the carrier model. Thus the previously reported anomaly in the exchange experiments was merely an apparent one due, first, to the fact that different sugars possess different maximum velocities and, second, to the accumulation of the unlabelled sugars within the cell.

Apparent desensitization of a σ receptor sub-population in neonatal rat cardiac myocytes by pre-treatment with σ receptor ligands

Abstract σ Receptor ligands induce marked effects on contractility in cardiac myocytes from neonatal and adult rats (Ela et al., 1994, J. Pharmacol. Exp. Ther. 269, 1300–1309; Novakova et al., 1995, Eur. J. Pharmacol. 286, 19–30). Augmentation or attenuation of the contractile amplitude was observed under different experimental conditions. Preincubation of neonatal cardiomyocytes with a σ receptor ligand ((+)-(3-hydroxyphenyl)-N-(1-propyl)-piperidine ((+)-3PPP), (+)-pentazocine, or haloperidol) changed the response to re-application of the ligand after cell wash. The inhibitory effect was abolished, while the stimulatory effect became much more pronounced. We suggest that the effects of σ receptor ligands are mediated via two receptor subtypes, one stimulatory and the other inhibitory, and only the inhibitory subtype is subject to desensitization.