Highly selective sigma 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 1S,2 R-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)-cycloh exylamine (BD-737) (0.1-100 nM) and N-[2-(3,4-dichlorophenyl)ethyl]-N,N',N'-trimethylethylenediamine (BD-1047) (0.01-10 nM), caused potentiation of electrically-evoked amplitudes of contraction and Ca2+ 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 Ca2+ 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.

Distinct components of morphine effects on cardiac myocytes are mediated by the kappa and delta opioid receptors.

Morphine exerts direct effects on cultured cardiac myocytes from neonatal rats. These effects are mediated via the delta and the kappa opioid receptors, as mu opioid receptors are not present in neonatal cardiomyocyte cultures. Binding parameters to the delta and kappa opioid receptors were determined in membrane preparations from these cultures by heterologous competition to [3H]diprenorphine binding, with [D-Pen2, D-Pen5]-enkephalin (DPDPE) and trans-(dl)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate (U-50,488H) as specific displacers respectively. To define the components of morphine effects mediated via activation of either the delta or the kappa opioid receptor alone, cardiac myocytes were exposed to morphine in the presence of specific antagonists to the kappa or delta opioid receptor respectively. Activation of the kappa opioid receptors by morphine caused a transient increase in Ca2+ influx, leading to increase in amplitudes of [Ca2+]i transients and contraction, with no change in the intracellular pH. Activation of the delta opioid receptors alone by morphine caused a decrease in the amplitude of contraction. This decrease was mediated by a decrease in the intracellular pH leading to reduced responsiveness of the myofilaments to Ca2+. There was no change in Ca2+ influx and in the amplitude of [Ca2+]i transients. The effects mediated through the delta opioid but not through the kappa opioid receptors were pertussis toxin sensitive, indicating coupling of the delta opioid receptors to pertussis toxin sensitive GTP-binding proteins. The overall effects of morphine on the neonatal cardiac myocytes were the sum of the effects exerted by morphine when it activated each of the opioid receptors alone.

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.

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

sigma 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 sigma 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 sigma receptor ligands are mediated via two receptor subtypes, one stimulatory and the other inhibitory, and only the inhibitory subtype is subject to desensitization.

Inotropic action of sigma 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.

Sigma receptor ligands modulate contractility, Ca++ influx and beating rate in cultured cardiac myocytes.

Specific binding of [3H]-1,3-di-o-tolylguanidine (DTG) and (+)-[3H]-(3-hydroxyphenyl)-N-(1-propyl)-piperidine [(+)-3-PPP] to membranes of cultured cardiac myocytes from neonatal rats revealed the presence of sigma receptors on these cells. Exposure of cultured cardiomyocytes to nanomolar concentrations of (+)-3-PPP, (+)-pentazocine and haloperidol induced specific patterns of changes in contractility of electrically paced cultures. The amplitude of systolic cell-motion (ASM) decreased by 10 to 25% 1 to 2 min after drug addition, then transiently increased (3-10 min) and finally decreased to about 75% of control level. Fluorescence measurements on indo-1 loaded cardiomyocytes revealed drug-induced changes in the size of the concentration of free cytosolic calcium ([Ca++]i)-transients, similar to the changes observed in ASM. These changes appear to be mediated by corresponding changes in the rates of 45Ca++ influx which increased 2 to 7 min after the addition of (+)-3-PPP and decrease to 50% of the control level thereafter. Preincubation with thapsigargin, which depletes the sarcoplasmic reticulum-Ca++ stores, did not affect the pattern of changes in ASM, induced by the subsequent addition of (+)-3-PPP. This indicates that the changes in [Ca++]i are not mediated by sarcoplasmic reticulum-Ca++ transport systems. Exposure to sigma ligands did not affect the apparent sensitivity of the myofilaments to Ca++, as indicated by the relationships between changes in ASM and in [Ca++]i-transients. Cultures which were not paced, contracted spontaneously at a constant rhythm. Sigma receptor ligands caused changes in beating frequencies which were followed by irregular contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Opioid effects on contractility, Ca(2+)-transients and intracellular pH in cultured cardiac myocytes.

Morphine, the opioid-agonist, and the antagonists naloxone and levallorphan exerted direct effects on spontaneously-contracting cultures of cardiac myocytes from neonatal rats. Naloxone and levallorphan induced an increase in the amplitude of systolic cell motion (ASM) and in the size of [Ca2+]i-transients, measured as indo-1 fluorescence ratio (IFR), whereas morphine caused an increase in IFR with no change in ASM. Both morphine and naloxone caused a transient increase in 45Ca2+ influx into the cardiomyocytes. Analysis of the relationship between changes in ASM and IFR indicated dual action of the drugs: (a) An increase in [Ca2+]i-transients elicited by morphine and the antagonists, apparently resulting from a transient increase of Ca2+ influx. (b) Altered myofibril responsiveness to Ca2+; the agonists decreased it, and the antagonists increased it. Intracellular pHi measurements in cardiomyocytes loaded with the fluorescent indicator BCECF revealed that morphine caused acidosis and the antagonists caused alkalosis. These pH changes were inhibited by pertussis-toxin, protein kinase inhibitor K323a, phorbol-ester and ethylisopropyl-amiloride, indicating pathways mediated by GTP-binding proteins and altered activities of protein kinase C and Na+/H+ exchanger. Preincubation with pertussis toxin prior to the addition of morphine prevented the decrease in the myofibril responsiveness to Ca2+ as well as the decrease in pHi but did not affect the increase in [Ca2+]i-transients and the increase in the rate of Ca2+ influx. As a result, addition of morphine after preincubation with pertussis toxin caused a positive inotropic effect. Our results indicate that morphine acts by two different pathways distinguishable by their sensitivity to pertussis toxin (1), increased Ca2+ influx leading to increased Ca(2+)-transients and (2) decreased intracellular pH leading to reduced myofibril responsiveness to Ca2+.

Induction of IL-4 secretion by the radiation leukemia virus (RadLV): role in autocrine growth stimulation of RadLV infected pre-leukemic cells.

Intrathymic inoculation of radiation-leukemia virus (RadLV) into C57BL/6 mice induces a population of pre-leukemic (PL) T cells which progress into clonal, mature thymic lymphomas after a latency period of 3 to 5 months. In order to understand how PL cells are retained in the thymus for a prolonged period of time we determined whether RadLV infected cells secrete and/or respond to a T-cell growth factor that may be involved in the long-term maintenance of a thymic PL-cell pool. We have previously found that in vitro proliferation of RadLV-infected PL cells is IL-4-dependent. Here we show that RadLV induces IL-4 secretion and IL-4 receptor (IL-4R) expression in normal thymic lymphocytes. RadLV-infected PL thymocytes express IL-4R and secrete IL-4. Their IL-4 secretion could be enhanced if incubated in the presence of RadLV and this enhancement was inhibited by anti-RadLV antibodies. Several RadLV-induced lymphoma lines secreted IL-4 and/or expressed IL-4R, but these features were not essential for their continuous growth. The results suggest that RadLV induces IL-4-dependent autocrine growth which maintains a population of PL T cells in the thymus. Transition from a PL state to overt thymic lymphoma involves emancipation of a PL cell from IL-4 dependency.