Wafer scale interdigitated nanoelectrode devices functionalized using a MEMS-based deposition system.

This paper reports on a methodology to elaborate interdigitated nanoelectrode devices (INDs) at the wafer scale, relying on a mix-and-match process which combines proximity optical lithography and electron beam lithography. An optimum exposure dose allowed fabricating nanodevices, at the wafer level, with a successful yield of 97%. The final devices are bonded onto conventional TO-8 packages. Electrical characterization in a short-circuited nanoelectrode is performed, revealing a 230 µΩ cm resistivity value at 23 °C. A MEMS-based spotter made of cantilevers (called Bioplume) has been used to obtain precise functionalization of the INDs with sub-picoliter volume solutions. These INDs are the basis of multiple tunnel junction nanodevices, intended to serve as novel highly sensitive nanobiosensors.

kappa-Opioid receptor-transfected cell lines: modulation of adenylyl cyclase activity following acute and chronic opioid treatments.

The opioid receptors mu, delta and kappa have recently been cloned. Here we show that kappa-agonists inhibit adenylyl cyclase activity in Chinese hamster ovary cells stably transfected with rat kappa-opioid receptor cDNA. Chronic exposure of the cells to kappa-agonists did not lead to significant desensitization of the capacity of the agonists to inhibit adenylyl cyclase. On the other hand, withdrawal of the agonist following the chronic treatment led to the phenomenon of supersensitivity ('overshoot') of adenylyl cyclase activity. Both the inhibition of adenylyl cyclase activity by the acute opioid treatment and the chronic agonist-induced supersensitivity are pertussis toxin sensitive, demonstrating involvement of Gi/Go proteins in both processes.

Increased expression of synapsin I mRNA in defined areas of the rat central nervous system following chronic morphine treatment.

Chronic opiate administration leads to a selective regulation of several cellular proteins and mRNAs. This phenomenon has been viewed as a compensatory mechanism to the opiate signaling leading to the development of opiate addiction. In this study, in situ hybridization histochemistry experiments were employed to investigate the effect of chronic morphine treatment on synapsin I gene expression. We show here for the first time that prolonged morphine exposure causes a selective increase in the mRNA levels of synapsin I in several brain regions which are considered to be important for opiate action. Quantitative analysis of the signals, obtained by hybridization of digoxigenin-labeled antisense RNA probe, revealed a 5.8- and 7-fold increase of synapsin I mRNA levels in the locus coeruleus and the amygdala of morphine-treated rats, respectively, as compared with control untreated rats. Increased expression of synapsin I mRNA was also observed in the spinal cord of morphine-treated rats (by 3.8-fold). Since opiates were shown to attenuate neurotransmitter release and reduce synapsin I phosphorylation, it is suggested that the increase in synapsin I levels would lead to the requirement of higher amounts of opiate agonists to obtain the opiate physiological effects. These results suggest that the increases in mRNA levels of synapsin I in these specific areas can be part of the molecular mechanism(s) underlying opiate tolerance and withdrawal.

Ras-GRF, the activator of Ras, is expressed preferentially in mature neurons of the central nervous system.

In rodents, the Ras-specific guanine-nucleotide exchange factor (Ras-GRF) is expressed in different areas of the brain and, at a reduced level, also in the spinal cord. No expression of the 140 kDa Ras-GRF was detected in dorsal root ganglia and all other tissues tested. Analysis of primary cultures derived from brain reveals that this exchange factor is only present in neurons of the central nervous system. In primary hippocampal cultures, the expression of Ras-GRF increases in parallel with the onset of a neuronal network and in the whole brain it increases sharply after birth.

Modulation of thymidine incorporation by kappa-opioid ligands in rat spinal cord-dorsal root ganglion co-cultures.

beta-Endorphin, met-enkephalin and several mu-selective opioid agonists were shown to decrease thymidine incorporation into DNA in various neural cell cultures. We now report that the kappa-selective opioid agonists U50488, U69593 and MR2034 modulate [3H]thymidine incorporation into DNA in rat spinal cord-dorsal root ganglion co-cultures. U50488 at 10 microM increased by 60% thymidine incorporation in 6-day-old cultures. The thymidine incorporation induced by U50488 was blocked by the kappa-selective antagonist nor-binaltorphimine, as well as by pertussis toxin and LiCl. U50488 treatment stimulated phosphatidylinositol turnover by three-fold compared with untreated controls. These findings suggest that kappa-opioid agonists modulate DNA synthesis in spinal cord-dorsal root ganglion co-cultures through a mechanism which involves pertussis toxin-sensitive GTP-binding proteins, as well as activation of phosphatidylinositol turnover.

Cannabinoids inhibit agonist-stimulated formation of inositol phosphates in rat hippocampal cultures.

The effect of cannabinoids on phosphoinositide metabolism stimulated by activation of muscarinic receptors, alpha 1-adrenoceptors or glutamate receptors was examined in rat hippocampal cultures. Carbachol stimulated phosphoinositide turnover by 5.5-fold over basal level, whereas glutamate and norepinephrine stimulated phosphoinositide turnover by 2-fold. Addition of cannabinoids, such as delta 8-tetrahydrocannabinol, delta 9-tetrahydrocannabinol or the psychoinactive cannabidiol inhibited formation of inositol phosphates evoked by carbachol, glutamate or norepinephrine by 55-90%. The cannabinoids alone only slightly inhibited the basal unstimulated formation of inositol phosphates. The inhibitory effect of the cannabinoids was dose-dependent and was achieved within the range of pharmacologically relevant concentrations. IC50 values for delta 8-tetrahydrocannabinol, delta 9-tetrahydrocannabinol and cannabidiol were 9.6 +/- 1.0, 9.7 +/- 0.3 and 7.9 +/- 0.4 microM, respectively. Pretreatment with pertussis toxin (100 ng/ml, 18 h) did not affect the carbachol-induced stimulation of phosphoinositide turnover or its inhibition by the cannabinoids. This suggests that the inhibition by the cannabinoids of the stimulated formation of inositol phosphates is not mediated through a pertussis toxin-sensitive GTP-binding protein nor through the known effect of the cannabinoids on adenylate cyclase inhibition.

Pre- and postnatal development of opiate receptor subtypes in rat spinal cord.

We have studied the developmental expression of opiate binding sites in the rat spinal cord at various prenatal and postnatal stages. For each developmental stage, we have compared the expression pattern of kappa receptors with that of mu and delta receptor subtypes. Both mu and kappa receptors appear relatively early during spinal cord ontogeny (from the 15th prenatal day), while delta sites are expressed later at the postnatal period (starting at the 1st postnatal day). The number of kappa sites predominates throughout the development (55-80% of total opiate sites) with two peaks of binding activity: one at the 20th gestational day, and the other around the 7th postnatal day. mu sites represent 20-38% of the total opiate receptor population with one peak of binding activity appearing at the 1st postnatal day. The densities of mu and kappa receptors at the adult stage are lower by 40-50% than the peak values observed at the early postnatal periods. The relative amounts of delta sites remain low throughout the ontogeny (4-8% of the total opiate sites). The binding properties of neonatal (1 day after birth) kappa sites (ligand binding affinities, regulation of agonist binding by guanosine triphosphate and various cations) are similar to those displayed by kappa receptors in adult spinal cord.

Expression and regulation of kappa opiate receptors in rat spinal cord-dorsal root ganglion cocultures.

We have been using rat spinal cord-dorsal root ganglion primary cocultures (SC-DG) as a model system for exploring K receptor regulation. During the first 10 days in culture, the total number of opiate receptors increased markedly, reaching a Bmax of 180 fmoles/mg protein for K sites and a Bmax of 50 fmoles/mg protein for mu sites. Following this period of development, the K and mu receptor number did not change significantly. No detectable delta sites were observed at any time of culture. The binding of [3H]diprenorphine to K sites was found to be saturable, of high affinity and stereospecific. After chronic agonist treatment of cultured cells, K receptors did not down-regulate, whereas more than 50% of the mu receptor sites did. Following chronic antagonist treatment, mu receptors were markedly up-regulated (260% of control), while K sites exhibited a weaker up-regulation response (160% of control). These data demonstrate that K opiate receptors are expressed at high concentrations in SC-DG cultures and that contrary to mu receptors in spinal cord and delta receptors in NG10815 cells, K binding sites are less susceptible to modulation following chronic agonist or antagonist treatment. This suggests that K receptors may be regulated by different control mechanisms.

Kappa-opiate agonists inhibit adenylate cyclase and produce heterologous desensitization in rat spinal cord.

The nature of the opiate modulation of adenylate cyclase following acute and chronic agonist exposure has been investigated in rat spinal cord. Using membranes of both adult rat spinal cord and spinal cord-dorsal root ganglion cocultures, we found that kappa-opiate receptors are negatively coupled to adenylate cyclase. The kappa-opiate agonists (e.g., U50488) inhibit significantly and dose-dependently the basal and the forskolin-stimulated cyclase activities, whereas mu and delta agonists are ineffective. The regulatory action is stereospecific and requires the presence of GTP. EGTA treatment of the plasma membranes abolished the effect of kappa-opiate agonists on the basal cyclase activity, and this inhibitory effect could not be restored by subsequent addition of Ca2+. The EGTA treatment did not affect the kappa agonist inhibition of the forskolin-stimulated cyclase. The results also show that following chronic exposure of cultured cells to etorphine or U50488, there is a loss of kappa agonist inhibition of the cyclase. Moreover, this desensitization process appears to be heterologous, because alpha 2-adrenergic agonists (e.g., clonidine or norepinephrine) and the muscarinic agonist (carbachol) exhibited significantly lower potency for inhibiting cyclase activity when compared to untreated cultures. This pattern of heterologous desensitization suggests that chronic exposure to kappa opiates leads to alterations in postreceptor regulatory components, possibly GTP-binding proteins.

Kappa opiate agonists inhibit Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. Involvement of a GTP-binding protein.

The aim of the present study has been to characterize the regulation by opiates of 45Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. We have demonstrated that K+-induced depolarization, in the presence of the Ca2+ channel agonist Bay K8644, stimulated Ca2+ influx (3-4-fold) via the dihydropyridine class of voltage-dependent Ca2+ channels. While mu and delta opiates had no effect, kappa opiate agonists (e.g. U50488, dynorphin) profoundly depressed the stimulated Ca2+ influx (86% inhibition at 100 microM U50488). The kappa agonist action was stereospecific and could be reversed by the opiate antagonist naloxone. The inhibition produced by kappa agonists was greatly diminished following pertussis toxin treatment, and this effect was accompanied by toxin-induced ADP-ribosylation of a 40-41-kDa protein. This suggests that kappa opiate receptors are negatively coupled to voltage-dependent Ca2+ channels, via a pertussis toxin-sensitive GTP-binding protein. Basal 45Ca2+ uptake, stimulated by adenylate cyclase activators (forskolin and cholera toxin), was potently inhibited by kappa opiates suggesting that, under conditions of neurohormonal stimulation of adenylate cyclase, kappa receptors are coupled to Ca2+ channels indirectly via the adenylate cyclase complex. In addition, cAMP-independent coupling pathways may also be involved.

Long-term opiate exposure leads to increase in synapsin I in rat spinal cord-dorsal root ganglion cocultures.

Cocultures of spinal cord and dorsal root ganglion cells contain relatively high concentrations of kappa-opiate receptors. We have previously shown that acute kappa-opiate agonist treatment reduces phosphorylation of synapsin I stimulated by depolarizing agents (such as 60 mM KCl). Here we show that prolonged opiate treatment increases the levels of synapsin I immunoreactivity in the cells. Several opiate agonists, such as U50488, ethylketocyclazocine, dynorphin, and [D-Ala2,D-Leu5]enkephalin, caused a 3.0-3.4-fold increase in the immunoreactive level of synapsin I. The effect of the kappa-agonist U50488 on the up-regulation of synapsin I was dose dependent and was blocked by the kappa-opiate antagonist norbinaltor-phimine. The results suggest that continued activation of opiate receptors by chronic agonist treatment up-regulates the levels of synapsin I. This increase in synapsin I could contribute to the development of tolerance to opiates.

Opiate receptor agonists regulate phosphorylation of synapsin I in cocultures of rat spinal cord and dorsal root ganglion.

Kappa opiate receptor agonists applied to cocultures of spinal cord and dorsal root ganglion neurons have been previously shown to inhibit voltage-dependent Ca2+ influx and adenylate cyclase activity. Here we describe the effect of kappa opiate receptor agonists on phosphorylation of synapsin I, a synaptic-vesicle-associated protein whose phosphorylation was shown to be regulated by cAMP and Ca2+ concentrations. Depolarization of spinal cord-dorsal root ganglion cocultured cells (by high K+ or veratridine) and the addition of forskolin (which activates adenylate cyclase) led to increased phosphorylation of synapsin I. Addition of kappa opiate agonists attenuated both the depolarization- and the forskolin-induced phosphorylation of synapsin I. This attenuation was blocked by the opiate antagonist naloxone. mu and delta opiate receptor agonists had much weaker effects on the depolarization-induced phosphorylation of synapsin I. Similarly, kappa opiate agonists decreased (by 40-60%) the high-K+- or veratridine-induced phosphorylation of synapsin I in spinal cord synaptosomes. These results show that opiate ligands modulate synapsin I phosphorylation. Moreover, the data could explain the reduction in synaptic efficacy observed after opiate treatment.

Opiate-induced adenylyl cyclase superactivation is isozyme-specific.

While acute activation of inhibitory Gi/o-coupled receptors leads to inhibition of adenylyl cyclase, chronic activation of such receptors leads to an increase in cAMP accumulation. This phenomenon, observed in many cell types, has been referred to as adenylyl cyclase superactivation. At this stage, the mechanism leading to adenylyl cyclase superactivation and the nature of the isozyme(s) responsible for this phenomenon are largely unknown. Here we show that transfection of adenylyl cyclase isozymes into COS-7 cells results in an isozyme-specific increase in AC activity upon stimulation (e.g. with forskolin, ionomycin, or stimulatory receptor ligands). However, independently of the method used to activate specific adenylyl cyclase isozymes, acute activation of the mu-opioid receptor inhibited the activity of adenylyl cyclases I, V, VI, and VIII, while types II, IV, and VII were stimulated and type III was not affected. Chronic mu-opioid receptor activation followed by removal of the agonist was previously shown, in transfected COS-7 cells, to induce superactivation of adenylyl cyclase type V. Here we show that it also leads to superactivation of adenylyl cyclase types I, VI, and VIII, but not of type II, III, IV, or VII, demonstrating that the superactivation is isozyme-specific. Not only were isozymes II, IV, and VII not superactivated, but a reduction in the activities of these isozymes was actually observed upon chronic opiate exposure. These results suggest that the phenomena of tolerance and withdrawal involve specific adenylyl cyclase isozymes.

Low doses of anandamides inhibit pharmacological effects of delta 9-tetrahydrocannabinol.

It has been shown previously that the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide 20:4, n-6) induces in vivo and in vivo effects typical of a cannabinoid partial agonist. We now report that the synthetic docosahexaenylethanolamide (anandamide 22:6, n-3) shows similar activities. In addition we show that these two anandamides, under certain experimental conditions, antagonize the effects of delta 9-THC both in vivo and in vitro. Thus a significant decrease in the potency of delta 9-THC-induced inhibition of adenylate cyclase was observed in N18TG2 neuroblastoma cells that were pretreated with low concentrations of anandamides. At these low concentrations of anandamides had no effect when applied alone. In vivo, Sabra or ICR mice were subjected to a tetrad of tests, designed to detect cannabinoid-induced effects. Mice pretreated (i.p.) with 10 mg/kg of delta 9-THC received injections with anandamides. Only low doses (0.0001-0.1 mg/kg) of the anandamides, which had no effects when administered alone, partially or fully inhibited the THC-induced effects. These findings suggest that the inhibition of delta 9-THC-induced effects by low doses of anandamides may be due to partial agonistic effects of these materials. It is possible that low doses of the anandamides are capable of activating a Gs protein mediated signaling pathway, or may cause an allosteric modulation of the cannabinoid receptor.

Anandamide, a brain endogenous compound, interacts specifically with cannabinoid receptors and inhibits adenylate cyclase.

A putative endogenous cannabinoid ligand, arachidonylethanolamide (termed "anandamide"), was isolated recently from porcine brain. Here we demonstrate that this compound is a specific cannabinoid agonist and exerts its action directly via the cannabinoid receptors. Anandamide specifically binds to membranes from cells transiently (COS) or stably (Chinese hamster ovary) transfected with an expression plasmid carrying the cannabinoid receptor DNA but not to membranes from control nontransfected cells. Moreover, anandamide inhibited the forskolin-stimulated adenylate cyclase in the transfected cells and in cells that naturally express cannabinoid receptors (N18TG2 neuroblastoma) but not in control nontransfected cells. As with exogenous cannabinoids, the inhibition by anandamide of the forskolin-stimulated adenylate cyclase was blocked by treatment with pertussis toxin. These data indicate that anandamide is an endogenous agonist that may serve as a genuine neurotransmitter for the cannabinoid receptor.

Opioids inhibit endothelin-mediated DNA synthesis, phosphoinositide turnover, and Ca2+ mobilization in rat C6 glioma cells.

Opioid agonists inhibit DNA synthesis in C6 rat glioma cells that express opioid receptors, induced by desipramine (DMI). This inhibition was not observed in cells that were not treated with DMI, and thus did not express opioid-binding sites. Endothelin, a known mitogen, increased thymidine incorporation dose dependently (up to 1.7-fold) in DMI-treated C6 cells. This increase was reversed by an anti-idiotypic antibody to opioid receptors, Ab2AOR, which has opioid agonist properties. The opioid antagonist naltrexone blocked the inhibition caused by Ab2AOR. Endothelin also stimulated phosphoinositide (PI) turnover and this effect was inhibited by morphine (50%) or by Ab2AOR (72%) in DMI-treated but not in DMI-untreated C6 cells. These actions of morphine and Ab2AOR were reversed by naltrexone. The inhibition of PI turnover and of thymidine incorporation by Ab2AOR or morphine was insensitive to pertussis toxin (PTX). Since PI turnover is known to induce Ca2+ mobilization, it was of interest to examine the effects of the applied opioids on intracellular Ca2+ concentrations. Endothelin increased the concentration of cytosolic free Ca2+ in the cells while Ab2AOR, morphine, and beta-endorphin reversed the endothelin-induced Ca2+ mobilization in DMI-treated but not in DMI-untreated C6 cells. The effect of these agonists was also blocked by naltrexone. The results indicate that glial cells can be a target of an opioid receptor-mediated antimitogenic action and that an abatement in PI turnover and Ca2+ mobilization may be associated with this mechanism.