Transient changes in four GLUT4 compartments in rat adipocytes during the transition, insulin-stimulated to basal: implications for the GLUT4 trafficking pathway.

In rat adipocytes, insulin-induced GLUT4 recruitment to the plasma membrane (PM) is associated with characteristic changes in the GLUT4 contents of three distinct endosomal fractions, T, H, and L. The organelle-specific marker distribution pattern suggests that these endosomal GLUT4 compartments are sorting endosomes (SR), GLUT4-storage endosomes (ST), and GLUT4 exocytotic vesicules (EV), respectively, prompting us to analyze GLUT4 recycling based upon a four-compartment kinetic model. Our analysis revealed that insulin modulates GLUT4 trafficking at multiple steps, including not only the endocytotic and exocytotic rates, but also the two rate coefficients coupling the three intracellular compartments. This analysis assumes that GLUT4 cycles through PM T, H,L, and back to PM, in that order, with transitions characterized by four first-order coefficients. Values assigned to these coefficients are based upon the four steady-state GLUT4 pool sizes assessed under both basal and insulin stimulated states and the transition time courses observed in the plasma membrane GLUT4 pool. Here we present the first reported experimental measurements of transient changes in each of the four GLUT4 compartments during the insulin-stimulated to basal transition in rat adipocytes and compare these experimental results with the corresponding model simulations. The close correlation of these results offers clear support for the general validity of the assumed model structure and the assignment of the T compartment to the sorting endosome GLUT4 pool. Variations in the recycling pathway from that of an unbranched cyclic topography are also considered in the light of these experimental observations. The possibility that H is a coupled GLUT4 storage compartment lying outside the direct cyclic pathway is contraindicated by the data. Okadaic acid-induced GLUT4 recruitment is accompanied by modulation of the rate coefficients linking individual endosomal GLUT4 compartments, further demonstrating a significant role of the endosomal pathways in GLUT4 exocytosis.

Hypothalamic CRH mRNA levels are differentially modulated by repeated 'binge' cocaine with or without D(1) dopamine receptor blockade.

We previously found that there was a rapid stimulatory effect of acute (1 day) 'binge' cocaine on CRH mRNA levels in the rat hypothalamus. In contrast, after 3 days of 'binge' cocaine, there was a modest decrease (12%) in hypothalamic CRH mRNA levels, which after 14 days of 'binge' cocaine was greater (32%) and significantly lower than control values. Also, our previous studies found an elevation of CRH mRNA in the frontal cortex after 3 days of 'binge' cocaine. The present study was designed to investigate the possible role of dopamine receptors in modulating these effects. Administration of 3 days of 'binge' cocaine (3 x 15 mg/kg, i.p.) was preceded by daily injections of either D(1) (SCH23390, 2 mg/kg) or D(2) (sulpiride, 50 mg/kg) dopamine receptor antagonist. Neither SCH23390 nor sulpiride had an effect on basal CRH mRNA levels in the hypothalamus, frontal cortex or amygdala. Small decreases (10-13%) in hypothalamic CRH mRNA levels were found again to be induced by 3 days of repeated 'binge' cocaine. However, this modest decrease was not found in the rats that received D(1) antagonist SCH23390 pretreatment. Pretreatment with D(2) antagonist sulpiride had no effect on this decrease. These findings suggest that the inhibitory effect of repeated 'binge' cocaine on the hypothalamic CRH mRNA expression is absent when there is D(1), but not D(2), dopamine receptor blockade. In the frontal cortex, pretreatment with either SCH23390 or sulpiride did not alter the increases in the CRH mRNA levels induced by repeated 'binge' cocaine. The results suggest that the cocaine-induced modulation of hypothalamic CRH mRNA expression is secondary to changes in the activity of specific components of dopaminergic systems.

Opposing gates model for voltage gating of gap junction channels.

Gap junctions are intercellular channels that link the cytoplasm of neighboring cells. Because a gap junction channel is composed of two connexons docking head-to-head with each other, the channel voltage-gating profile is symmetrical for homotypic channels made of two identical connexons (hemichannels) and asymmetric for the heterotypic channels made of two different connexons (i.e., different connexin composition). In this study we have developed a gating model that allows quantitative characterization of the voltage gating of homotypic and heterotypic channels. This model differs from the present model in use by integrating, rather than separating, the contributions of the voltage gates of the two member connexons. The gating profile can now be fitted over the entire voltage range, eliminating the previous need for data splicing and fusion of two hemichannel descriptions, which is problematic when dealing with heterotypic channels. This model also provides a practical formula to render quantitative several previously qualitative concepts, including a similarity principle for matching a voltage gate to its host connexon, assignment of gating polarity to a connexon, and the effect of docking interactions between two member connexons in an intact gap junction channel.

Elevation of guinea pig brain preprodynorphin mRNA expression and hypothalamic-pituitary-adrenal axis activity by "binge" pattern cocaine administration.

The endogenous opioid system and the hypothalamic-pituitary-adrenal (HPA) axis have been implicated in many of the neurobiological effects of cocaine. Previous studies in our laboratory showed that "binge" pattern cocaine administration increases preprodynorphin (ppDyn) mRNA levels in the caudate putamen and circulating levels of corticosterone in the rat. The present study extended these findings to guinea pigs, a species known to have a kappa opioid receptor profile similar to that of humans. Male guinea pigs were treated with: (a) "binge" pattern cocaine for 7 days (subchronic) (3 x 15 mg/kg/day, hourly, intraperitoneal); (b) "binge" pattern saline for 5 days followed by "binge" pattern cocaine for 2 days (subacute); or (c) "binge" pattern saline for 7 days. Thirty minutes after the final injection, levels of ppDyn mRNA were quantitated in the nucleus accumbens, caudate putamen, frontal cortex, amygdala, hippocampus, and hypothalamus using a solution hybridization RNase protection assay. Regional distribution of ppDyn mRNA levels in the guinea pig brain was similar to that found in rat, with highest levels in the nucleus accumbens and caudate putamen. In the caudate putamen, ppDyn mRNA was significantly increased following either 2 days (38% increase) or 7 days (32% increase) of "binge" pattern cocaine administration as compared to saline-treated controls. No significant changes in ppDyn mRNA levels were found in any other brain region. Both subacute and subchronic "binge" cocaine administration significantly elevated plasma levels of adrenocorticotropin hormone (ACTH) and cortisol. However, the ACTH and cortisol increases were significantly blunted following 7 days of "binge" cocaine administration as compared to 2 days of drug treatment, reflecting the development of HPA tolerance or adaptation to repeated cocaine administration. Thus, the ppDyn mRNA and HPA responses to cocaine in guinea pigs are similar to those observed in rats.

Stem vascular architecture in the rattan palm Calamus (Arecaceae-Calamoideae-Calaminae).

Climbing stems in the rattan genus Calamus can reach lengths of well over 100 m, are long-lived, and yet their vascular tissue is entirely primary. Such a combination suggests that stem vasculature is efficient and resistant to hydraulic disruption. By means of an optical shuttle and video recording of sequential images we analyzed the stem of a cultivated species. The stem has vascular features that are unusual compared with those in arborescent palms and seemingly inefficient in terms of long-distance water transport. Axial bundles are discontinuous basally because leaf traces, when followed downwards, always end blindly below. Furthermore, there is no regular distal branching of each leaf trace at its level of departure into a leaf, so that neither a continuing axial bundle nor bridges to adjacent axial bundles are produced as in the standard palm construction. Instead, the axial bundles in the stem periphery are connected to leaf traces and to each other by narrow and irregular transverse or oblique commissures that are not the developmental homologues of bridges. As in other palms, metaxylem within a leaf trace is not continuous into the leaf so that the only connection to a leaf is via protoxylem. Within the stem, protoxylem (tracheids) and metaxylem (vessels) are never contiguous, unlike in other palms, which suggests that water can only move from metaxylem to protoxylem, and hence into the leaf, across a hydraulic resistance. We suggest that this minimizes cavitation of vessels and/or may be associated with an unknown mechanism that refills embolized vessels. Also, the metaxylem can be significant in stem water storage in the absence of abundant ground parenchyma.

Adenosine and adenosine triphosphate modulate the substrate binding affinity of glucose transporter GLUT1 in vitro.

Evidence indicates that a large portion of the facilitative glucose transporter isoform GLUT1 in certain animal cells is kept inactive and activated in response to acute metabolic stresses. A reversible interaction of a certain inhibitor molecule with GLUT1 protein has been implicated in this process. In an effort to identify this putative GLUT1 inhibitor molecule, we studied here the effects of adenosine and adenosine triphosphate (ATP) on the binding of D-glucose to GLUT1 by assessing their abilities to displace cytochalasin B (CB), using purified GLUT1 in vesicles. At pH 7.4, adenosine competitively inhibited CB binding to GLUT1 and also reduced the substrate binding affinity by more than an order of magnitude, both with an apparent dissociation constant (K(D)) of 3.0 mM. ATP had no effect on CB and D-glucose binding to GLUT1, but reduced adenosine binding affinity to GLUT1 by 2-fold with a K(D) of 30 mM. At pH 3.6, however, ATP inhibited the CB binding nearly competitively, and increased the substrate binding affinity by 4--5-fold, both with an apparent K(D) of 1.22 mM. These findings clearly demonstrate that adenosine and ATP interact with GLUT1 in vitro and modulate its substrate binding affinity. They also suggest that adenosine and ATP may regulate GLUT1 intrinsic activity in certain cells where adenosine reduces the substrate-binding affinity while ATP increases the substrate-binding affinity by interfering with the adenosine effect and/or by enhancing the substrate-binding affinity at an acidic compartment.

Detection of single nucleotide polymorphisms of the human mu opioid receptor gene by hybridization or single nucleotide extension on custom oligonucleotide gelpad microchips: potential in studies of addiction.

The human mu opioid receptor (MOR) plays a central role in mediating the effects of opioids, both endogenous and exogenous. Epidemiological studies have shown that addiction in general, and especially opiate addiction, has a heritable component. Clinical and laboratory studies suggest that the MOR gene may contribute to the heritable component of vulnerability to develop opiate addiction. Naturally occurring single nucleotide polymorphisms (SNPs) have been identified in the MOR gene by conventional methods. Two coding region SNPs, the A118G and C17T substitutions, occur at high allelic frequencies (10.5% and 6.6%, respectively, in our previous studies). These common SNPs cause amino acid changes in the receptor, and may have implications for differences in individual responses to opioids, as well as decreased or increased vulnerability to opiate addiction. The A118G substitution encodes a variant receptor with binding and signal transduction differences in response to beta-endorphin in cellular assays. Recent innovations in microchip technology offer new potential methods for SNP detection. We report here on the development of two separate approaches using custom oligonucleotide gelpad microarrays for detection of these two common SNPs of the MOR gene in human DNA samples. First, PCR-amplified genomic DNA samples were used to produce target sequences, which were labeled with fluorescent dye and hybridized to custom microchips. Oligonucleotides on these reusable microchips were designed to query nucleotide substitutions at positions 17 and 118 of the MOR gene. Thirty-six human DNA samples were assayed both on these custom microchips and by conventional automated gel sequencing, with highly concordant identification of both heterozygous and homozygous substitutions. A second approach was developed for the C17T SNP utilizing single nucleotide extension on custom microchips. These custom gelpad microchips have potential for the rapid and inexpensive detection of specific SNPs for genetic and genomic studies.

Reduced hypothalamic POMC and anterior pituitary CRF1 receptor mRNA levels after acute, but not chronic, daily "binge" intragastric alcohol administration.

BACKGROUND: Endogenous corticotropin-releasing factor (CRF), its pituitary CRF1 receptor, and proopiomelanocortin (POMC) may be involved in the hypothalamic-pituitary-adrenal (HPA) responses to alcohol. METHODS: Alcohol (1.5 g/kg) or water was administered intragastrically to male Fischer rats after the "binge" pattern regimen, that is, three times daily at 1 hr intervals at the beginning of the light cycle. The levels of CRF, CRF1 receptor, and POMC mRNAs in the hypothalamic-pituitary axis were measured after acute (1 day) or chronic (14 days) binge pattern alcohol administration. Plasma levels of ACTH and corticosterone were measured to examine time-dependent alterations of HPA responses. RESULTS: Plasma ACTH and corticosterone levels were elevated dramatically after 1 day of acute binge pattern alcohol administration. After 14 days of chronic alcohol, however, no elevation in plasma ACTH levels and an attenuated elevation in plasma corticosterone levels were found. CRF mRNA levels in the hypothalamus were not altered after either acute or chronic alcohol administration. CRF1 receptor mRNA levels in the anterior pituitary were decreased significantly after acute administration, with no change after chronic alcohol administration. POMC mRNA levels in the anterior pituitary were not altered by either acute or chronic alcohol administration. In the hypothalamus, POMC mRNA levels were decreased significantly after acute but not chronic binge alcohol administration. CONCLUSIONS: These results suggest that (1) rats exposed to chronic binge alcohol develop tolerance in HPA activity, as shown by no elevation of ACTH and an attenuated corticosterone response to chronic alcohol after initial dramatic elevations by acute alcohol administration; (2) a concurrent acute decrease in CRF1 receptor mRNA levels in the anterior pituitary is associated with increased HPA activity, and (3) alterations of POMC gene expression in the hypothalamic region may have implications for a molecular understanding of the neuroendocrine response to alcohol.

Modulation of GLUT4 and GLUT1 recycling by insulin in rat adipocytes: kinetic analysis based on the involvement of multiple intracellular compartments.

The trafficking kinetics of GLUT4 and GLUT1 in rat epididymal adipocytes were analyzed by a four-compartment model based upon steady-state pool sizes of three intracellular fractions and one plasma membrane fraction separated and assessed under both basal and insulin-stimulated states. The steady-state compartment sizes provided relative values of the kinetic coefficients characterizing the rate of each process in the loop. Absolute values of these coefficients were obtained by matching the simulated half-times to those observed experimentally and reported in the literature for both basal and insulin-stimulated states. Our analysis revealed that insulin modulates the GLUT4 trafficking at multiple steps in the rat adipocyte, not only reducing the endocytotic rate constant 3-4-fold and increasing the exocytotic rate 8-24-fold but also increasing the two rate coefficients coupling the three intracellular compartments 2-6-fold each. Furthermore, GLUT1 was completely segregated from GLUT4 in two of the three intracellular compartments, and its steady-state distribution is consistent with a four-compartment model of GLUT1 recycling involving an insulin sensitive endocytosis step in common with the GLUT4 system, but with all other processes being insensitive to insulin.

Hypothalamic-pituitary-adrenal activity and pro-opiomelanocortin mRNA levels in the hypothalamus and pituitary of the rat are differentially modulated by acute intermittent morphine with or without water restriction stress.

Acute administration of morphine stimulates the secretion of hypothalamic-pituitary-adrenal (HPA) hormones, ACTH, beta-endorphin and corticosterone in the rat. In this study we investigated the effects of repeated multiple-dose morphine on HPA activity under two different conditions: without or with water restriction stress. Rats received six intermittent injections of morphine (6.25 mg/kg per injection, s.c.) every 2 h and were killed 30 min after the last injection. The results were as follows. (1) Morphine significantly elevated plasma ACTH and corticosterone levels; water restriction also significantly increased ACTH secretion, but with no significant increase of plasma corticosterone levels. In contrast, rats treated with morphine under the water restriction condition failed to show any increases of either ACTH or corticosterone levels. (2) Morphine did not change pro-opiomelanocortin (POMC) mRNA levels in the anterior pituitary; whereas water restriction significantly increased the POMC mRNA levels. The water restriction-induced increases of POMC mRNA in the anterior pituitary were absent in the rats which received morphine. (3) Morphine significantly increased POMC mRNA levels in the hypothalamus; water restriction had no effect. The morphine-induced increases in POMC mRNA in the hypothalamus were absent in the rat under the water restriction condition. These findings, that the effects of morphine on HPA activation or POMC mRNA expression depend on the presence of stress, suggest a counter-regulatory role of opiates on a stress response and opioid gene expression.

Effects of chronic 'Binge' cocaine administration on plasma ACTH and corticosterone levels in mice deficient in DARPP-32.

The product of the DARPP-32 gene mediates intracellular signals initiated by the binding of dopamine to its receptors. Cocaine administration leads to increased activation of dopamine receptors, and causes activation of the stress-responsive hypothalamic-pituitary-adrenal (HPA) axis. We determined the effects of chronic 'binge' pattern cocaine on HPA activity in mice containing a targeted disruption of the DARPP-32 gene. Mice received three daily injections of cocaine (15 mg/kg/injection) for 14 days, and were sacrificed 30 min after the last injection. We measured the levels of plasma adrenocorticotropin (ACTH) and corticosterone which reflect HPA activity. In wild-type controls, 'binge' cocaine administration significantly increased plasma ACTH and corticosterone levels. In contrast, DARPP-32-deficient mice failed to show a significant elevation of either plasma ACTH or corticosterone levels following 'binge' cocaine. The results indicate that DARPP-32 plays a role in mediating the stimulatory effects of cocaine on the HPA axis.

Acute "binge" cocaine increases mu-opioid receptor mRNA levels in areas of the rat mesolimbic mesocortical dopamine system.

Autoradiography studies demonstrated that chronic "binge" cocaine administration increased mu-opioid receptor density in dopaminergically innervated rat brain regions, including the cingulate cortex, the nucleus accumbens, and the basolateral amygdala. The present study investigated the effects of a single day of binge-pattern cocaine administration (3 x 15 mg/kg, intraperitoneally [i.p.] at hourly intervals) on mu-opioid receptor mRNA levels in selected brain regions. Rats were sacrificed 30 min after the third injection and mRNA levels were measured by a quantitative solution hybridization RNase protection assay. Acute binge cocaine administration significantly increased mu-opioid receptor mRNA levels in the frontal cortex, nucleus accumbens, and amygdala, but not in the caudate-putamen, thalamus, hippocampus, and hypothalamus. As has been suggested for other G-protein coupled receptors, the rapid increase of MOR mRNA reported in this study might represent an adaptive response to compensate for a decrease in number of receptors following cocaine-induced opioid peptide release.

Acute intermittent morphine increases preprodynorphin and kappa opioid receptor mRNA levels in the rat brain.

We determined the effects of morphine on mRNA levels for the opioid ligands preprodynorphin (PPD) and preproenkephalin (PPE) and the kappa opioid receptor (KOR). Rats received six injections of morphine (6.25 mg/kg/injection) every 2 h, and were sacrificed 30 min later. mRNA levels were measured in brain tissue after removal of the cortex, cerebellum and brainstem. There were increases in PPD and KOR mRNA levels (P<0.05 and P<0.005, respectively), with no alteration of PPE. These alterations in the kappa/dynorphin system may counter morphine-induced effects on the brain.

Effects of memantine alone and with acute 'binge' cocaine on hypothalamic-pituitary-adrenal activity in the rat.

The effects of memantine, a non-competitive NMDA-receptor antagonist used in the management of dementia, and its coadministration with acute 'binge' pattern cocaine on hypothalamic-pituitary-adrenal axis activity were investigated in the rat. Measurements 3 h after injections showed that memantine alone at 20 mg kg(-1) (i.p.), but not 10 mg kg(-1), increased corticotropin-releasing factor (CRF) mRNA levels in the hypothalamus and both adrenocorticotropic hormone and corticosterone levels in the blood, and decreased type I CRF receptor mRNA in the anterior pituitary. Our previous studies have shown that acute 'binge' cocaine increases CRF mRNA levels in the hypothalamus. In this study, pretreatment with memantine (10 and 20 mg kg(-1), i.p.) did not alter the up-regulation of hypothalamic CRF mRNA induced by acute 'binge' cocaine (3 x 15 mg kg(-1), i.p.). Of interest, pretreatment with memantine at 10 mg kg(-1), which alone had no effect on corticosterone levels, caused a greater elevation of corticosterone levels in combination with 'binge' cocaine than acute 'binge' cocaine alone, indicating that memantine does not attenuate 'binge' cocaine-stimulated hypothalamic-pituitary-adrenal activity. These results indicate that both memantine and acute 'binge' cocaine stimulate hypothalamic-pituitary-adrenal activity by activating CRF neurons in the hypothalamus.

Behavioral stereotypies induced by "binge' cocaine administration are independent of drug-induced increases in corticosterone levels.

Cocaine administration causes dramatic stereotypic behavior and elevation of circulating corticosterone levels in rodents. The present study tested the possible role of increased corticosterone in mediating stereotypic behavior caused by "binge' pattern cocaine administration. Animals were administered saline or cocaine intraperitoneally for 3 days, with or without pretreatment with a D1 (SCH 23390, 2 mg/kg) or D2 (sulpiride, 50 mg/kg) dopamine receptor antagonist. Three days of cocaine "binges' significantly increased corticosterone levels in vehicle pretreated rats (P < 0.01). Both SCH 23390 and sulpiride pretreatment daily significantly attenuated this increase (P < 0.01). Cocaine administration caused stereotypic behaviors in vehicle pretreatment rats (P < 0.01). These behavioral responses were blocked by the D1 dopamine receptor antagonist SCH 23390, but not by the D2 antagonist sulpiride. These findings reaffirm the dominant role of the D1 receptor in mediating behavioral stereotypy caused by elevations of extracellular dopamine in the synaptic cleft. The fact that the dose of sulpiride used in these studies prevented the elevation of plasma corticosterone caused by cocaine, without blocking the stereotypy caused by cocaine, indicates that this stereotypic behavior does not require drug-induced elevation in circulating levels of corticosterone.

Chronic food restriction and streptozotocin-induced diabetes differentially alter prodynorphin mRNA levels in rat brain regions.

It was previously reported that chronic food restriction and streptozotocin-induced diabetes lead to brain region-specific changes in levels of Prodyn-derived peptides. These changes parallel behavioral adaptations that are reversed by opioid antagonists. In the present study, effects of food restriction and diabetes on Prodyn gene expression were measured in rat brain regions using a quantitative solution hybridization mRNA assay. Picogram amounts of Prodyn mRNA were determined in extracts of five brain regions. The highest density of Prodyn mRNA was observed in extracts of nucleus accumbens (4.68 pg/microg total RNA), bed nucleus of the stria terminalis (4.18 pg/microg), and in caudate nucleus (3.51 pg/microg). Lower levels were observed in the lateral hypothalamus (1.87 pg/microg) and central nucleus of the amygdala (1.22 pg/microg). Food restriction and diabetes both markedly increased the levels of Prodyn mRNA in the central amygdala (163% and 93%, respectively). Levels in the lateral hypothalamus were also increased (35% and 29%, respectively), though only the food-restriction effect was statistically significant. Neither treatment altered prodynorphin mRNA levels in the caudate nucleus, nucleus accumbens or bed nucleus of the stria terminalis. These results suggest that dynorphin neurons in central amygdala and lateral hypothalamus may be involved in behavioral or physiological adaptations to sustained metabolic need.

Quantitation of dopamine transporter mRNA in the rat brain: mapping, effects of "binge" cocaine administration and withdrawal.

Dopamine transporter (DAT) mRNA from selected brain regions of individual male Fischer rats was quantitated utilizing a sensitive solution hybridization assay in which the levels of RNase-protected 32P-labeled mRNA:cRNA hybrids were measured. DAT mRNA was detected in whole brain regions known to contain abundant DAT mRNA (mean picogram of DAT mRNA/microgram of total RNA +/- SEM): substantia nigra, 7.17 +/- 0.47; ventral tegmentum, 4.71 +/- 0.38. In regions known to contain low levels of DAT mRNA, these levels were detected: central grey, 0.39 +/- 0.06; hypothalamus, 0.14 +/- 0.03. In addition, DAT mRNA was detected in areas where it had not previously been identified: amygdala, 0.19 +/- 0.03; caudate-putamen, 0.15 +/- 0.03; nucleus accumbens, 0.13 +/- 0.01; pons/medulla, 0.12 +/- 0.02; globus pallidus, 0.09 +/- 0.04; pituitary 0.07 +/- 0.01; frontal cortex, 0.05 +/- 0.01. No DAT mRNA was detected in 150 micrograms of rat liver RNA. As cocaine binds to and inhibits the activity of the dopamine transporter, we sought to determine if there were differences in dopamine transporter mRNA levels between saline- and cocaine-injected rats or rats withdrawn from a chronic "binge" pattern (15 mg/kg per dose i.p.; three doses at 1 h intervals each day) cocaine injection. Using trichloroacetic acid precipitation of mRNA:cRNA hybrids from RNA extracted from whole brain regions, we found no significant differences in the substantia nigra or the ventral tegmentum following subacute (3 days) binge, chronic (14 days) binge or 10 days withdrawal from a chronic binge pattern cocaine or saline administration.

Prodynorphin, proenkephalin and kappa opioid receptor mRNA responses to acute "binge" cocaine.

Previous studies showed that preprodynorphin (ppDyn) mRNA increases in caudate-putamen while kappa opioid receptor (KOR) mRNA decreases in substantia nigra after 3 and 14 days "binge" cocaine. To further characterize opioid mRNA responses, rats were administered: saline; 1 day cocaine followed by 1 day saline; 1 day cocaine; or 2 days cocaine. ppDyn mRNA in caudate-putamen increased in both groups receiving cocaine on the final day compared to groups receiving saline. Preproenkephalin (ppEnk) mRNA in caudate-putamen increased, and KOR mRNA in substantia nigra decreased, after 2 days of cocaine. Thus ppDyn mRNA is elevated acutely by cocaine, while ppEnk and KOR mRNAs show a significant response only on the second day of "binge" cocaine.

Guinea pig preprodynorphin mRNA: primary structure and regional quantitation in the brain.

We isolated and sequenced genomic and cDNA clones of the guinea pig preprodynorphin (ppDyn) mRNA. The sequence of ppDyn mRNA was deduced from a combination of genomic and cDNA clones: The primary structure of two coding exons was derived from a genomic clone and 5' and 3' untranslated sequences were obtained using rapid amplification of cDNA ends (RACE). The predicted mRNA of 2,350 nucleotides coincides well with the size of transcripts in Northern blot analyses of RNA from different brain regions. The deduced amino acid sequence of guinea pig ppDyn shares 70%, 68%, and 61% identity to porcine, human, and rat ppDyn, respectively. The 5' untranslated sequences of guinea pig hippocampal and adrenal ppDyn mRNA are identical; both contain sequences of exon I and, like porcine mRNA, lack an exon (exon II) present in human and rat mRNA. Quantitative solution hybridization RNase protection analysis of total RNA from selected guinea pig brain regions was performed. The nucleus accumbens was found to have the greatest abundance of ppDyn mRNA, followed by caudate putamen, hippocampus, hypothalamus, amygdala, frontal cortex, olfactory bulb, and pons/medulla.