Regulation of sigma-1 receptors and endoplasmic reticulum chaperones in the brain of methamphetamine self-administering rats.

sigma-1 Receptors are endoplasmic reticulum (ER) chaperones that are implicated in the neuroplasticity associated with psychostimulant abuse. We immunocytochemically examined the distribution of sigma-1 receptors in the brain of drug-naive rats and then examined the dynamics of sigma-1 receptors and other ER chaperones in specific brain subregions of rats that self-administered methamphetamine, received methamphetamine passively, or received only saline injections. sigma-1 Receptors were found to be expressed in moderate to high levels in the olfactory bulb, striatum, nucleus accumbens shell, olfactory tubercle, amygdala, hippocampus, red nucleus, ventral tegmental area, substantia nigra, and locus ceruleus. Methamphetamine, whether self-administered or passively received, significantly elevated ER chaperones including the sigma-1 receptor, BiP, and calreticulin in the ventral tegmental area and substantia nigra. In the olfactory bulb, however, only the sigma-1 receptor chaperone was increased, and this increase occurred only in rats that actively self-administered methamphetamine. Consistent with an increase in sigma-1 receptors, extracellular signal-regulated kinase was found to be activated and protein kinase A attenuated in the olfactory bulb of methamphetamine self-administering rats. sigma-1 Receptors in the olfactory bulb were found to be colocalized with dopamine D1 receptors. These results indicate that methamphetamine induces ER stress in the ventral tegmental area and substantia nigra in rats whether the drug is received actively or passively. However, the changes seen only in rats that actively self-administered methamphetamine suggest that D1 and sigma-1 receptors in the olfactory bulb might play an important role in the motivational conditioning/learning aspects of methamphetamine self-administration in the rat.

Compartmentalization of cardiac beta-adrenergic inotropy modulation by phosphodiesterase type 5.

BACKGROUND: Recent cell-based studies have found that cGMP synthesis and hydrolysis by phosphodiesterase (PDE) appear compartmentalized, with nitric oxide synthase-derived and/or PDE type 5 (PDE-5)-hydrolyzable cGMP undetected at the sarcolemmal membrane in contrast to cGMP stimulated by natriuretic peptide. In the present study, we determine the functional significance of such compartments with a comparison of beta-adrenergic modulation by PDE-5 inhibition to that of natriuretic peptide stimulation in both cardiomyocytes and intact hearts. The potential role of differential cGMP and protein kinase G stimulation by these 2 modulators was also studied. METHODS AND RESULTS: Intact C57/BL6 mouse hearts were studied with pressure-volume analysis, and adult isolated myocytes were studied with fluorescence microscopy. PDE-5 inhibition with 0.1 to 1 micromol/L sildenafil (SIL) suppressed isoproterenol (ISO)-stimulated contractility, whereas 10 micromol/L atrial natriuretic peptide (ANP) had no effect. ISO suppression by SIL was prevented in cells pretreated with a protein kinase G inhibitor. Surprisingly, myocardial cGMP changed little with SIL+ISO yet rose nearly 5-fold with ANP, whereas protein kinase G activation (vasodilator-stimulated protein phosphorylation; ELISA assay) displayed the opposite: increased with SIL+ISO but unaltered by ANP+ISO. PDE-5 and ANP compartments were functionally separated, as inhibition of nitric oxide synthase by N(w)-nitro-L-arginine methyl ester eliminated antiadrenergic effects of SIL, yet this was not restorable by co-stimulation with ANP. CONCLUSIONS: Regulation of cardiac beta-adrenergic response by cGMP is specifically linked to a nitric oxide-synthesis/PDE-5-hydrolyzed pool signaling via protein kinase G. Natriuretic peptide stimulation achieves greater detectable increases in cGMP but not protein kinase G activity and does not modulate beta-adrenergic response. Such disparities likely contribute to differential cardiac regulation by drugs that modulate cGMP synthesis and hydrolysis.

Protein kinase A activation down-regulates, whereas extracellular signal-regulated kinase activation up-regulates sigma-1 receptors in B-104 cells: Implication for neuroplasticity.

The sigma-1 receptor (Sig-1R) can bind psychostimulants and was shown to be up-regulated in the brain of methamphetamine self-administering rats. Up-regulation of Sig-1Rs has been implicated in neuroplasticity. However, the mechanism(s) whereby Sig-1Rs are up-regulated by psychostimulants is unknown. Here, we employed a neuroblastoma cell line B-104, devoid of dopamine receptors and transporter, and examined the effects of psychostimulants as well as cAMP on the expression of Sig-1Rs in this cell line, with a specific goal to identify signal transduction pathway(s) that may regulate Sig-1R expression. Chronic treatments of B-104 cells with physiological concentrations of cocaine or methamphetamine failed to alter the expression of Sig-1Rs. N6,2'-O-Dibutyryl-cAMP (dB-cAMP), when used at 0.5 mM, caused a down-regulation of Sig-1Rs that could be blocked by a protein kinase A (PKA) inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89). However, dB-cAMP, when used at 2 mM, caused an up-regulation of Sig-1Rs that was insensitive to the H-89 blockade but was partially blocked by an extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone). Furthermore, 2 mM dB-cAMP induced an ERK phosphorylation lasting at least 90 min, at which time the phosphorylation caused by 0.5 mM dB-cAMP had already diminished. PD98059, applied 90 min after addition of 2 mM dB-cAMP, attenuated the Sig-1R up-regulation. Our results indicate that cAMP is bimodal in regulating Sig-1R expression: a down-regulation via PKA and an up-regulation via ERK. Results also suggest that psychostimulants may manipulate the cAMP-PKA-Sig-1R and/or the cAMP-ERK-Sig-1R pathways to achieve a neuroplasticity that favors addictive behaviors.

Peroxynitrite and myocardial contractility: in vivo versus in vitro effects.

Generation of peroxynitrite (ONOO-) as a result of altered redox balance has been shown to affect cardiac function; however, inconsistencies in the data exist, particularly for myocardial contractility. The hypothesis that the cardiac impact of ONOO- formation depends on its site of generation, intravascular or intramyocardial, was examined. Cardiac contractility was assessed by pressure-volume analysis to delineate vascular versus cardiac changes on direct infusion of ONOO- into the right atria of conscious dogs both with normal cardiac function and in heart failure. Additionally, ONOO- was administered to isolated murine cardiomyocytes to mimic in situ cardiac generation. When infused in vivo, ONOO- had little impact on inotropy but led to systemic arterial dilation, likely as a result of rapid decomposition to NO2- and NO3-. In contrast, infused ONOO- was long lived enough to abolish beta-adrenergic (dobutamine)-stimulated contractility/relaxation, most likely through catecholamine oxidation to aminochrome. When administered to isolated murine cardiomyocytes, ONOO- induced a rapid reduction in sarcomere shortening and whole cell calcium transients, although neither decomposed ONOO- or NaNO2 had any effect. Thus, systemic generation of ONOO- is unlikely to have primary cardiac effects, but may modulate cardiac contractile reserve, via blunted beta-adrenergic stimulation, and vascular tone, as a result of generation of NO2- and NO3-. However, myocyte generation of ONOO- may impair contractile function by directly altering Ca2+ handling. These data demonstrate that the site of generation within the cardiovascular system largely dictates the ability of ONOO- to directly or indirectly modulate cardiac pump function.

Effect of growth factors on DNA labeling and cytoskeletal protein expression in 17-beta-estradiol and basic fibroblast growth factor pre-treated astrocyte cultures.

Astrocytes react to all noxae which damage neurons. Their reactions include degeneration, hypertrophy, hyperplasia and fibre formation. Growth factors inducing proliferation and differentiation of both neurons and astrocytes in culture play a pivotal role in the dynamic flow of signaling molecules between neurons and astroglia. Estrogens as well influence astroglia and are neuroprotectants. This study has investigated the interactions between growth factors and estrogens on DNA labeling and cytoskeletal protein [glial fibrillary acidic protein (GFAP) and vimentin] expression in 22 DIV astrocyte cultures treated for 24 or 36 h under different experimental conditions. Contemporary addition of 17-beta-estradiol (E2) with two or three growth factors for 24 h, significantly stimulated methyl-[3H]thymidine incorporation into DNA from 22 days in vitro (DIV) astrocyte cultures. This effect reached a peak when E2 was co-added with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and insulin. In astrocyte cultures treated for 36 h with E2 and EGF + insulin or bFGF + insulin added in the last 12 h, DNA labeling was remarkably increased. The parallel cyclin Dl expression positively correlated with ERK2 activation. Western blot analysis for cytoskeletal proteins showed also changes of both GFAP and vimentin expression. The above data suggest the occurrence of a scheduled interaction between "competence" or "progression" growth factors and estrogens on DNA labeling and cytoskeletal protein expression during astroglial cell proliferation and differentiation in culture. A better understanding of the mechanisms of these interactions may contribute to develop strategies for controlling astroglial reaction in cerebrovascular disease including stroke and hypertensive brain damage.

Effect of growth factors on nuclear and mitochondrial ADP-ribosylation processes during astroglial cell development and aging in culture.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and insulin (INS) are powerful mitogens and may regulate gene expression in cultured astrocytes by ADP-ribosylation process. Nuclear poly-ADP ribose polymerase (PARP) and mitochondrial monoADP-ribosyltransferase (ADPRT) are the key enzymes involved in poly-ADP-ribosylation and mono ADP-ribosylation, respectively. In this investigation the effect of EGF, bFGF, IGF-I or INS on nuclear PARP and mitochondrial ADPRT activities were assessed in nuclei and mitochondria purified from developing (30 DIV) or aging (90 and 190 DIV) primary rat astrocyte cultures. A marked increase of PARP activity in bFGF or IGF-I treated astroglial cell cultures at 30 DIV was found. Nuclear PARP and mitochondrial ADPRT activities were greatly stimulated by treatment with EGF or INS alone or together in astrocyte cultures at 30 DIV. Nuclear PARP and mitochondrial ADPRT activities showed a more remarkable increase in control untreated astrocyte cultures at 190 DIV than at 90 DIV. These findings suggest that ADP-ribosylation process is involved in DNA damage and repair during cell differentiation and aging in culture. Twelve hours treatment with EGF, INS or bFGF significantly stimulated nuclear PARP and mitochondrial ADPRT activities in 190 DIV aging astrocyte cultures. The above results indicate that EGF, INS and bFGF may play a crucial role in the post-translational modification of chromosomal proteins including ADP-ribosylation process in in vitro models. This suggests that growth factors regulate genomic stability in glial cells during development and maturation, stimulating nuclear and mitochondrial ADP-ribosylation processes in developing or aging astrocyte cultures.