TLN-4601 peripheral benzodiazepine receptor (PBR/TSPO) binding properties do not mediate apoptosis but confer tumor-specific accumulation.

TLN-4601 is a farnesylated dibenzodiazepinone isolated from Micromonospora sp. with an antiproliferative effect on several human cancer cell lines. Although the mechanism of action of TLN-4601 is unknown, our earlier work indicated that TLN-4601 binds the PBR (peripheral benzodiazepine receptor; more recently known as the translocator protein or TSPO), an 18 kDa protein associated with the mitochondrial permeability transition (mPT) pore. While the exact function of the PBR remains a matter of debate, it has been implicated in heme and steroid synthesis, cellular growth and differentiation, oxygen consumption and apoptosis. Using the Jurkat immortalized T-lymphocyte cell line, documented to have negligible PBR expression, and Jurkat cells stably transfected with a human PBR cDNA, the present study demonstrates that TLN-4601 induces apoptosis independently of PBR expression. As PBRs are overexpressed in brain tumors compared to normal brain, we examined if TLN-4601 would preferentially accumulate in tumors using an intra-cerebral tumor model. Our results demonstrate the ability of TLN-4601 to effectively bind the PBR in vivo as determined by competitive binding assay and receptor occupancy. Analysis of TLN-4601 tissue and plasma indicated that TLN-4601 preferentially accumulates in the tumor. Indeed, drug levels were 200-fold higher in the tumor compared to the normal brain. TLN-4601 accumulation in the tumor (176 µg/g) was also significant compared to liver (24.8 µg/g; 7-fold) and plasma (16.2 µg/mL; 11-fold). Taken together our data indicate that while PBR binding does not mediate cell growth inhibition and apoptosis, PBR binding may allow for the specific accumulation of TLN-4601 in PBR positive tumors.

Modulation of the contact hypersensitivity response by AE-941 (Neovastat), a novel antiangiogenic agent.

BACKGROUND: AE-941 (Neovastat) is an angiogenesis inhibitor noted to have antiinflammatory properties. OBJECTIVE: We tested Neovastat in a contact hypersensitivity (CHS) model to determine the mechanism of action of its antiinflammatory effects. METHODS: Neovastat was orally administered (200 mg/kg/day) during the sensitization and challenge phases of a murine CHS assay and inflammatory responses were measured. Subsequent assays were performed on mice treated with Neovastat or Cortisone (120 mg/kg/day, IP) and differential mRNA expression of several pro- and antiinflammatory cytokines was quantified using RT-PCR. RESULTS: Neovastat decreased inflammation by 39% when administered during sensitization but did not alter the CHS response when given during the challenge phase. Neovastat significantly induced IL-10 expression in skin and skin-draining lymph nodes (49% and 45%, respectively) and decreased IFNgamma expression in the lymph nodes (35%). CONCLUSION: Antiinflammatory effects of Neovastat observed in CHS could be linked to modulation of cytokines early in the sensitization phase.

Neovastat, a naturally occurring multifunctional antiangiogenic drug, in phase III clinical trials.

Recent studies have indicated that bone marrow angiogenesis is increased in multiple myeloma, suggesting that treatment with an antiangiogenic agent might be useful. Among the new antiangiogenic drugs in development, Neovastat (AE-941; Aeterna Laboratories, Quebec City, Canada) can be classified as a naturally occurring multifunctional antiangiogenic agent. It has a marked inhibitory effect on the formation of blood vessels in the chicken embryo vascularization assay (EVT) and endothelial cell proliferation. Furthermore, in vivo experiments showed that oral administration of Neovastat blocks the formation of blood vessels in Matrigel implants containing basic fibroblast growth factor (bFGF). The antiangiogenic activity of Neovastat was found to be associated with two mechanisms of action. In addition to the inhibition of the matrix metalloproteinase activities (MMP-2, MMP-9, and MMP-12), Neovastat inhibits vascular endothelial growth factor (VEGF) binding to endothelial cells, VEGF-dependent tyrosine phosphorylation, and VEGF-induced vascular permeability in mice. Neovastat was also found to have a significant antitumor activity. Oral administration of Neovastat in mice with subcutaneous grafted breast cancer (DA3) cells showed a significant reduction in tumor volume. Neovastat also decreased the number of lung metastases in the Lewis lung carcinoma model. Interestingly, the effect of Neovastat was additive to cisplatin in this model. Furthermore, no treatment-related mortality or loss of body weight was observed. Also, toxicology studies in rats and monkeys demonstrate no dose-limiting toxicity or target organ damage after 1 year of chronic exposure, thus suggesting that Neovastat could be safely administered in humans. Four clinical studies have been conducted to establish the dosing, safety, and early efficacy of Neovastat administered orally. In the oncology field, 482 patients have received Neovastat, of which 146 with solid tumors were exposed to the drug for more than 6 months. Two phase III clinical trials are currently underway. A phase III double-blind placebo-controlled study is being conducted to evaluate the efficacy of Neovastat in addition to induction chemotherapy/radiotherapy combined modality treatment in patients with unresectable non-small cell lung cancer stage IIIA and IIIB. A second phase III randomized, double-blind placebo-controlled study evaluates the efficacy of Neovastat as a monotherapy in metastatic renal cell carcinoma patients who have progressed following a first-line immunotherapy. Neovastat efficacy is also being evaluated in a registration phase II trial in patients with early relapse or refractory multiple myeloma.

Autoreceptor preference of dopamine D2 receptor agonists correlates with preferential coupling to cyclic AMP.

Dopamine autoreceptors control the synaptic release and turnover of dopamine. Some dopamine agonists display a preference for modulation of autoreceptor functions rather than postsynaptic-driven behaviors. However, the nature of this apparent selectivity is still elusive. To investigate this property, we have used an heterologous expression system in which D2S receptors are coupled to both inhibition of cyclic AMP levels and stimulation of inositol triphosphate production. We show that D2-like receptor agonists display distinct potencies on these two second messenger pathways. Moreover, a strong correlation is observed between the potency of agonists to interact with adenylate cyclase and their potency to modulate autoreceptor functions. Such a correlation does not show up with the phospholipase C pathway. This suggests that autoreceptor preference of D2-like receptor agonists may be driven by a preferential interaction with a second messenger system.

Dopamine D1 receptor protein is elevated in nucleus accumbens of human, chronic methamphetamine users.

Animal data have long suggested that an adaptive upregulation of nucleus accumbens dopamine D1 receptor function might underlie part of the dependency on drugs of abuse. We measured by quantitative immunoblotting protein levels of dopamine D1 and, for comparison, D2 receptors in brain of chronic users of methamphetamine, cocaine, and heroin. As compared with the controls, brain dopamine D1 receptor concentrations were selectively increased (by 44%) in the nucleus accumbens of the methamphetamine users, whereas a trend was observed in this brain area for reduced protein levels of the dopamine D2 receptor in all three drug groups (-25 to -37%; P < 0.05 for heroin group only). Our data support the hypothesis that aspects of the drug-dependent state in human methamphetamine users might be related to increased dopamine D1 receptor function in limbic brain.

Cellular colocalization of dopamine D1 mRNA and D2 receptor in rat brain using a D2 dopamine receptor specific polyclonal antibody.

1. The main objective of this work was to investigate the extent of cellular colocalization of dopamine D1 and D2 receptors in the rat brain. A double labeling technique, that combined immunocytochemical labeling of the D2 receptor using polyclonal antibodies raised against the third intracellular loop of the short isoform of the human D2 receptor in combination with in situ hybridization detecting D1 mRNA expression, was designed to accomplish this goal. 2. The specificity of the antisera obtained was confirmed by immunoprecipitation assay, Western blot analysis, and immunocytochemistry on D2R transfected cells and murine brain tissue. Western blot using the D2 receptor antibody revealed a specific broad band centered at 67 kDa in transfected cells and a major protein of 88 kDa corresponding to D2R expressed in the caudate-putamen, to a lesser extent in the cortex, and not at all detected in the hypothalamic region. 3. The content of neurons double-labeled for D1/D2 receptors was observed at in differing intensities in the dorsal endopiroform nucleus, the intercalated nucleus of amygdala, the anterior part of the cortical nucleus amygdala, the nucleus of the lateral olfactory tract, the piriform cortex, the parabrachial nucleus, the supraoptic nucleus and the parabigeminal nucleus. All other regions of the brain revealed neurons expressing either D1 or D2 dopamine receptors but not both at that same time. 4. These results clearly demonstrated that specific neurons expressed both receptors D1 and D2, and that this colocalization was restricted to particular regions of the rat brain.

Regulation by chronic treatment with cabergoline of dopamine D1 and D2 receptor levels and their expression in the striatum of Parkinsonian-monkeys.

1. Chronic treatment for one month with the long-acting dopamine D2-like agonist cabergoline (0.25 mg/kg s.c. every 48 hours), had despite partial tolerance, sustained antiparkinsonian activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonian monkeys (Macaca fascicularis). 2. Cabergoline treatment decreased by half striatal D2 receptor binding density measured by [3H]spiperone autoradiography versus untreated MPTP monkeys. No change in D2 mRNA measured by in situ hybridization and D2 receptor immunostaining was observed. 3. No change in either D1 receptor binding density or D1 receptor mRNA levels was observed in cabergoline-treated MPTP-monkeys compared to untreated MPTP-monkeys, suggesting receptor subfamily specificity of cabergoline. 4. The present results suggest that the cabergoline-induced behavioral partial tolerance is accompanied by a decrease in D2 receptor binding but not due to alterations in the steady state of D2 mRNA levels.

Veno-venous continuous renal replacement therapy for burned patients with acute renal failure.

From 1995 to 1998, 12 burned patients with acute renal failure (ARF) were treated by veno-venous continuous renal replacement therapy (CRRT) at the Burn Unit of Hôtel-Dieu de Montréal. Their mean (+/-SD) age was 51+/-12 years, and the mean burned surface covered 48.6+/-15.8% of total body surface area. All patients were mechanically ventilated and presented evidence of sepsis. The mean delay before occurrence of ARF was 15+/-6 days and ARF was mainly related to sepsis and hypotension. Main reasons for CRRT initiation were azotemia and fluid overload. A total of 15 CRRT modalities were applied (12 continuous veno-venous hemodiafiltration, CVVHDF; two continuous veno-venous hemofiltration, CVVH; and one continuous veno-venous hemodialysis, CVVHD) over 14+/-13 days. For CRRT, nine patients received heparin and three were not anticoagulated. Mean values for dialysate and reinjection flow rates were 1134+/-250 ml/h and 635+/-327 ml/h, respectively. Admission weight was 78.8+/-12.7 kg with a mean weight gain before CRRT initiation of 10.0+/-5.8 kg and a mean weight loss during CRRT of 8.9+/-5.5 kg. Nine patients received enteral plus parenteral nutrition, and three, parenteral nutrition only; the total caloric intake was 31.5+/-7.0 kcal/kg/day and protein intake, 1.8+/-0.4 g/kg/day. The normalized protein catabolic rate (nPCR) was evaluated at 2.28+/-0.78 g/kg/day during CRRT. The mortality rate was 50%. The six survivors all recovered normal renal function with four of them requiring intermittent hemodialysis for short periods. In conclusion, veno-venous CRRT is particularly well suited for this selected population allowing smooth fluid removal and aggressive nutritional support.

Characterization and distribution of Hxt1, a Na(+)/Cl(-)-dependent orphan transporter, in the human brain.

Rxt1, a transporter-like protein structurally related to the large family of Na(+)/Cl(-)-dependent carriers, was isolated from the rat brain. In the present study, Hxt1, the homologue of Rxt1, was isolated from human cortex cDNA. Comparison of their respective nucleotidic sequences revealed a 96% conservation between Hxt1 and Rxt1. Genetic mapping with human genome radiation hybrids allowed the location of the gene coding for Hxt1 between 323ya5 and 084xb3 AFM markers, on a portion of chromosome 1p which spans over 7 cM or 118 cRay. Northern blot analyses demonstrated that Hxt1 mRNA ( approximately 7.5 Kb) is expressed in the human brain but not in peripheral tissues. The immunodistribution of Hxt1 was determined with antibodies raised against the C-terminus of Rxt1. Hxt1 is concentrated in the cerebral cortex, caudate-putamen, substantia nigra, hippocampus, and cerebellum, appearing as a diffuse or a punctate labeling at the light microscope level. This regional and cellular distribution suggests that Hxt1, as its rat homologue, could be present in axon terminals of glutamatergic neurons. The high pressure of selection exerted upon this protein, its strategic anatomical and subcellular distributions suggest that this orphan transporter could be involved in critical functions in the central nervous system.

Neurotensin receptors and dopamine transporters: effects of MPTP lesioning and chronic dopaminergic treatments in monkeys.

The effect of denervation with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) of the dopamine (DA) nigrostriatal pathway on neurotensin (NT) receptor and DA transporter (DAT) in basal ganglia of monkeys (Macaca fascicularis) was investigated. The MPTP lesion induced a marked depletion of DA (90% or more vs. control) in the caudate nucleus and putamen. The densities of NT agonist binding sites labeled with [125I]NT and the NT antagonist binding sites labeled with [3H]SR142948A decreased by half in the caudate-putamen of MPTP-monkeys. In addition, the densities of [125I]NT and [3H]SR142948A binding sites markedly decreased (-77 and -63%, respectively) in the substantia nigra of MPTP-monkeys. Levocabastine did not compete with high affinity for [125I]NT binding in the monkey cingulate cortex, suggesting that only one class of NT receptors was labelled in the monkey brain. An extensive decrease of [3H]GBR12935 DAT binding sites (-92% vs. Control) was observed in the striatum of MPTP-monkeys and an important loss of DAT mRNA(-86% vs. Control) was observed in substantia nigra. Treatments for 1 month with either the D1 agonist SKF-82958 (3 mg/kg/day) or the D2 agonist cabergoline (0.25 mg/kg/day) had no effect on the lesion-induced decrease in NT and DAT binding sites or DAT mRNA levels. The decrease of striatal NT binding sites was less than expected from the decrease of DA content in this nucleus, suggesting only partial localization of NT receptors on nigrostriatal DAergic projections. These data also suggest that under severe DA denervation, treatment with D1 or D2 DA agonists does not modulate NT receptors and DAT density.

Continuous or pulsatile chronic D2 dopamine receptor agonist (U91356A) treatment of drug-naive 4-phenyl-1,2,3,6-tetrahydropyridine monkeys differentially regulates brain D1 and D2 receptor expression: in situ hybridization histochemical analysis.

The effect of a chronic D2 dopamine receptor agonist (U91356A) treatment on dopamine receptor gene expression in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys was investigated using quantitative in situ hybridization histochemistry. U91356A was administered to MPTP-monkeys for 27 days in a pulsatile (n=3) or continuous (n=3) schedule. Animals treated in a pulsatile mode showed progressive sensitization and developed dyskinesia; whereas with the continuous mode behavioural tolerance was observed but no dyskinesia developed. Untreated MPTP as well as naive control animals were also studied. The efficacy and uniformity of the MPTP effect was assessed by measures of dopamine concentrations by high performance liquid chromatography with electrochemical detection in the relevant brain areas. D1 and D2 receptor messenger RNAs levels were examined by in situ hybridization histochemistry using human complementary RNA probes. Intense specific labelling for D1 and D2 receptor messenger RNAs was measured in the caudate and putamen with a rostrocaudal gradient for D2 receptors and a lower density in the cortex for D1 receptors messenger RNA. D1 receptor mRNA levels in rostral striatum and cortex decreased whereas D2 receptor messenger RNA in caudal striatum increased in MPTP-monkeys compared to control animals. Continuous administration of U91356A reversed the MPTP-induced increase of D2 receptor messenger RNA, whereas the pulsatile administration did not significantly correct these messenger RNA changes. U91356A treatment whether continuous or pulsatile partially corrected the D1 receptor messenger RNA lesion-induced decrease in the striatum, whereas no correction was observed in the cortex. All MPTP-monkeys were extensively and similarly denervated suggesting that the D1 and D2 receptor expression changes following U91356A administration were treatment related. Our data show a lesion-induced imbalance of D1 (decrease) and D2 (increase) receptor messenger RNAs in the striatum of MPTP-monkeys. The response of these receptors to D1 agonist treatment showed receptor selectivity and was influenced by the time-course of drug delivery. Hence chronic continuous but not pulsatile administration of U91356A reversed the striatal D1 receptor messenger RNA increase.

Changes of D1 and D2 dopamine receptor mRNA in the brains of monkeys lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine: correction with chronic administration of L-3,4-dihydroxyphenylalanine.

The effect of L-3,4-dihydroxyphenylalanine (L-DOPA) on dopamine receptor gene expression in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys was investigated using in situ hybridization histochemistry with measures of changes in relative absorbance. In MPTP-lesioned monkeys, a decrease of D1 dopamine receptor mRNAs was observed in the rostral part of the caudate and putamen compared with control animals (-20% and -17%, respectively, in the lateral axis). Chronic treatment of MPTP-lesioned monkeys with L-DOPA returned their D1 receptor mRNA values to near those of control monkeys in the caudate and putamen (92% and 91% of control values, respectively). No lesion or drug-induced changes of D1 receptor mRNAs were observed in the more caudal parts of the striatum. A decrease of D1 receptor mRNAs was observed in the olfactory tubercule (-22%) in MPTP-lesioned monkeys compared with control animals but no change was seen in the nucleus accumbens. D1 receptor mRNAs in the anterior cerebral cortex were decreased in MPTP-lesioned monkeys (-19% compared with control animals). D1 receptor mRNAs in olfactory tubercle and in cerebral cortex of L-DOPA-treated MPTP-lesioned monkeys were not significantly different from control animals. For D2 receptor mRNAs, we observed an increase in the caudal part of the caudate and putamen (+24% and +23%, respectively, in MPTP-lesioned monkeys compared with control animals). Chronic L-DOPA treatment corrected this elevation to control values. No variation of D2 receptor mRNAs was seen in the more rostral parts of the striatum and in the nucleus accumbens in MPTP-lesioned monkeys as well as in MPTP-lesioned monkeys treated chronically with L-DOPA. Our results show for the first time that L-DOPA can influence gene expression of D1 and D2 receptors in MPTP-lesioned monkeys and correct the lesion-induced increase in the expression of D2 receptors, whereas the correction of the D1 receptor expression decrease is only partial. Furthermore, the changes in gene expression of D1 and D2 receptors in MPTP-lesioned monkeys are regional: they are restricted to the anterior striatum for the D1 receptors and the posterior striatum for the D2 receptors.

Eicosanoid modulation of the norepinephrine effect on blood pressure and renal hemodynamics in humans.

The main objective of this study was to investigate the role of eicosanoids in modulating the effect of norepinephrine (NE) on blood pressure and renal hemodynamics during NE administration. Eight healthy volunteers were randomly assigned to three (1 week apart) infusion periods (180 min) with either dextrose 5% or NE, with or without indomethacin pretreatment. Pressor doses of NE induced marked alterations in renal hemodynamics and concomitant increases in eicosanoid excretion rates. The production of the vasodilatory prostacyclin (PGI2), as reflected in the excretion rate of the stable metabolites 6-keto-prostaglandin (PG)F1(alpha) and 2,3-dinor-6-keto-PGF1(alpha), was 2.7 times higher than that of the constrictor thromboxane (TX)A2, which was measured as the stable derivative TXB2. Indomethacin pretreatment blunted the NE-induced augmentation in eicosanoid excretion and resulted in further increases in arterial pressure and in renal vascular resistance. These results demonstrate that PGI2 attenuates the systemic and the renal hemodynamic vasoconstrictor effect of NE in normotensive control normal subjects.

Brain dopamine transporter: gender differences and effect of chronic haloperidol.

Gender differences and the effect of chronic haloperidol on the rat brain dopamine transporter is reported. The density of striatal dopamine transporter sites labelled with [3H]GBR 12935, and of substantia nigra dopamine transporter mRNA measured by in situ hybridization were higher in female compared to male rats whereas striatal D2 specific binding labelled with [3H]spiperone was not significantly higher. Daily haloperidol treatment (1 mg/kg, i.p.) for 21 days increased striatal [3H]spiperone specific binding but left unchanged striatal [3H]GBR 12935 binding density and affinity as well as substantia nigra dopamine transporter mRNA levels. A reduce clearance rate of dopamine in the striatum after acute and chronic haloperidol was previously reported; the present results indicate that this may occur without changes in the sites of dopamine transport or in gene expression of this transporter.

Pituitary PRL secretion induced by tetraethylammonium is inhibited by dopamine through D2 receptors.

We have evaluated the inhibitory effect of dopamine on PRL secretion induced by blocking K+ channels. Tumor-derived GH4C1 cells and collagenase-dispersed normal anterior pituitary (AP) cells from young adult male rats were perifused with Krebs-Ringer Hepes medium. In both cell types blocking K+ channels with tetraethylammonium (TEA) induced PRL secretion but did not stimulate cyclic AMP generation. Blocking Na+ channels with 1 microM tetrodotoxin had no effect on basal or TEA-induced PRL secretion. Dopamine inhibited the TEA-induced rise in [Ca2+]i in GH4C1 cells expressing dopamine D2 short receptors. In normal AP cells, 1-100 nM dopamine blocked PRL secretion induced by 20 mM TEA in a log-linear concentration-dependent fashion, with a plateau at > 100 nM dopamine (IC50 30 nM). The D2 dopaminergic receptor agonist, quinpirole, at 100 nM completely blocked PRL secretion induced by 20 mM TEA. The D2 dopaminergic receptor antagonist, sulpiride, at 10 microM reversed the inhibitory effect of 10 microM dopamine on PRL secretion induced by 20 mM TEA. Pretreatment of cells with 100 ng/ml pertussis toxin (PTX) for 24 h prevented 100 nM dopamine inhibition of PRL secretion induced by 20 mM TEA. The data indicate that in both normal lactotroph cells and in tumor-derived cells expressing D2 receptors, PRL secretion stimulated by blocking K+ channels is inhibited by dopamine binding to D2 receptors on the plasma membrane. This inhibition involves interaction with PTX-sensitive Gi protein.

Recombination of endogenous D2 dopamine receptor gene with a metallothionein promoter in GH4C1 cells confers functional and inducible D2 response.

We have previously shown that expression of a functional endogenous D2 short dopamine receptor is obtained in GH4C1 cells following transfection with a plasmid that confers resistance to neomycin (pRSVNeo) (Allard et al. (1993) Biochem. Biophys. Res. Commun. 193, 801-807). In order to better understand the mechanisms responsible for such a phenomenon, we cloned and sequenced the 5' region of the D2 gene present in native GH4C1 cells as well as the cDNA of transfected cells. No homology with the published sequence of the rat D2 dopamine receptor promoter was found; however, this region has perfect homology with the mouse metallothionein promoter. In cells expressing D2 receptor, the promoter is fully functional and can regulate dopaminergic D2 receptor mRNA levels and receptor expression in a dose-dependent manner in the presence of Zn2+ or Cd2+. The receptor level is raised from 500 to 3000 fmol/mg of protein in the presence of 100 microM of Zn2+. These results suggest that in GH4C1 cells, a recombination between the mouse metallothionein promoter and the D2 dopamine receptor took place. This system provides us with a cell line expressing an endogenous dopamine D2 receptor in which the level of expression can be easily modulated.

Epidermal growth factor promotes uncoupling from adenylyl cyclase of the rat D2S receptor expressed in GH4C1 cells.

In anterior pituitary cells or when transfected into host cell lines, the D2 dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH-3 pituitary tumor cell line, which lacks functional D2 receptors, responds to epidermal growth factor (EGF) by expressing a D2 receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in alpha subunit of the G protein Gi3. In this study we have investigated the effects of EGF on the transduction mechanisms of D2 receptors in GH4C1 cells transfected and permanently overexpressing the rat short D2 receptor. Activation of D2 receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP-dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D2 receptor to inhibit cyclic AMP-dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of Gi3 protein. These results suggest that the transmembrane signaling specificity of G protein-coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.

Norepinephrine in neonatal rat ventricular myocytes: association with the cell nucleus and binding to nuclear alpha 1- and beta-adrenergic receptors.

Chronic exposure of neonatal rat ventricular myocytes to norepinephrine (NE) has been demonstrated to induce fetal cardiac gene expression and hypertrophy. The precise signaling mechanism of NE induction, as well as the long delay for the onset of NE effect, are not well understood. To examine the possibility that the hormone may be transported into the cell and exerts its effect through an intracellular site, ventricular myocytes from neonatal rats were incubated with [3H]-labeled NE and the cytosolic and nuclear fractions of the cell were measured for radioactivity. The presence of intracellular adrenergic binding sites was also explored. Following incubation of neonatal rat ventricular myocytes with [3H]NE for different time intervals (from 30 min to 22 h), the highest proportion (more than 80%) of NE taken up by the cell was recovered in the nuclear fraction. The nuclear accumulation was slow and time-dependent, being non-detectable in the first 60 min. Furthermore, isolated nuclei from the ventricular myocytes contain binding sites for [3H]prazosin and dihydroalprenolol, suggesting the presence of alpha 1 and beta 1 adrenergic receptors. The apparent KD and Bmax were 0.6 nM and 0.6 fmol/mg protein for alpha 1-adrenergic receptors, while beta-adrenergic nuclear receptors exhibited an apparent KD of 12 nM and a Bmax of 61 fmol/mg protein. Thus, neonatal rat ventricular myocytes exposed to NE accumulate the hormone in the cell nucleus where it can bind to high affinity alpha 1- and beta-adrenergic receptors.