Processing of proTRH to its intermediate products occurs before the packing into secretory granules of transfected AtT20 cells.

The intracellular compartments where posttranslational processing of proTRH takes place have not been identified. Using AtT20 cells transfected with a complementary DNA for preproTRH, we have used purified antibodies that recognize the intact precursor, intermediate and end products of processing to identify the subcellular compartments in which cleavage occur. Further, pulse-chase experiments followed by subcellular fractionation were undertaken to determine the order of processing of proTRH during its transport to the secretory granules. Cells were homogenized by nitrogen cavitation and subjected to a centrifugation of 1.065 mg/ml density gradient of Percoll to separate secretory granules (SG) from rough endoplasmic reticulum (RER)/Golgi apparatus. The purity of the SG and RER fractions was assessed by assays of marker enzymes for mitochondria, RER, Golgi, and cytoplasm. ProTRH derived cryptic peptides and TRH in each fraction were determined by RIA. Golgi and SG fractions were subjected to polyacrylamide gel electrophoresis followed by extraction and RIA. Using the anti-pCC10 antiserum which recognizes intact (26 kd) as well as partially processed prohormone, the RER/Golgi fraction contained 0.3 pmol intact ProTRH and 0.2 pmol each 15 and 6 kilodalton (kDa) fragments; the SG contained the 15 kDa moiety (0.2 pmol) along with a 6 kDa (0.4 pmol) material but not the 26 kDa ProTRH. The SG were also enriched by 0.21 pmol pYE27 (PreproTRH 25-50), 0.23 pmol pFT (PreproTRH 53-74), 0.31 pmol pEH24 (PreProTRH 86-106), and 0.5 pmol TRH. None of these were present in the RER/Golgi. Pulse-chase studies also showed that the intact proTRH (26 kDa) precursor was only present in the RER/Gg fraction along with two of its N-terminal intermediate processing products, a 15 k mol wt peptide and a 6 k mol wt peptide, and two of its C-terminal processing products, a 16.5 k mol wt and a 9.6 k mol wt peptides. In addition, fully processed peptides as well as TRH were only detected in the neurosecretory granules. These observations suggest that after the initial conversion of proTRH in the RER/Golgi fraction, the peptides are delivered to the granules where processing to TRH and cryptic peptides takes place. Supporting this, our pulse-chase studies unequivocally showed that, pEH24, an end product of proTRH processing, was only produced in secretory granules. Thus, initial cleavage of the TRH precursor may be required for packing and sorting of the end products to occur.

Identification of the thyrotropin-releasing hormone-prohormone and its posttranslational processing in a transfected AtT20 tumoral cell line.

By using an AtT20 cell line transfected with complementary DNA for preproTRH, we have identified the proTRH polyeptide precursor [26 kilodaltons (kDa)] and shown that this molecule gives rise to the proTRH derived sequences as determined by pulse-chase and trypsinization studies. The predicted proTRH precursor composed of 231 amino acids with 5 copies of a TRH progenitor sequence (Gln-His-Pro-Gly) and 7 other cryptic peptides was demonstrated by: 1) Western Blot analysis of an AtT20 cell extract with anti-pCC10 antibodies (an antibody that recognizes the intact prohormone as well as some intermediate products of processing); 2) Immunoprecipitation of the radiolabelled 26 kDa protein with anti-pCC10 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis; 3) Gel filtration chromatography of the radiolabeled 26 kDa extracted from SDS-PAGE. 4) RIA with anti-pCC10 antiserum against peptides extracted from adult rat hypothalamus and olfactory lobe after SDS-PAGE. 5) Trypsinization of the proTRH precursor which generated the proTRH cryptic peptides preproTRH25-50 (pYE27) and preproTRH53-74 (pFT22). These moieties were also produced during trypsinization of intermediate products of processing. By means of pulse-chase studies, the 26 kDa polypeptide was shown to be the biosynthetic precursor to all the proTRH derived cryptic peptides. Cleavage at two positions in the center of the molecule (Lys107-Arg108 and Lys152-Arg153) generated two moieties of 16.5 and 15 kDa. The 15 kDa N-terminal fragment is later cleaved to a 6 kDa peptide that includes the proTRH derived peptides, pYE27, pFT22, and pEH24. The carboxy-terminal 16.5 kDa fragment of the prohormone is processed to a 9.6 kDa fragment which contains the proTRH derived peptide pST10 (preproTRH160-169) and a fragment of 5.4 kDa that may be the C-terminal peptide preproTRH208-255 recognized by antisera pAC12 and pYE17. In further processing, the 9.6 kDa molecule is cleaved to produce a 5.4 kDa peptide from either sequences 115-169 or 160-199.

An anti-chi-1 antibody recognizes a heavily glycosylated protein in rat brain.

The chi(-1) subunit is a recently identified member of a new class of the ionotropic glutamate receptor family that attenuates NMDA receptor current. We have generated a polyclonal C-terminal antibody to the chi(-1) subunit which recognizes a 135-kDa protein in membranes prepared from chi(-1) transfected HEK-293 cells and in rat brain. In the post-natal day 7 (P7) rat brain, Western blot analysis revealed a 135-kDa band in the thalamus and cortex but not the striatum, cerebellum or peripheral tissues. De-glycosylation of the chi(-1) subunit in both transfected cell lines and in the brain reduced the 135-kDa band to 110 kDa, near the predicted molecular weight of the chi(-1) subunit. These studies demonstrate the chi(-1) subunit is expressed as a glycosylated protein subunit in a distribution that parallels that observed for chi(-1) mRNA by in situ hybridization.

Comparison of intravenous cocaethylene and cocaine in humans.

RATIONALE: Cocaethylene is a pharmacologically active homolog and metabolite of cocaine, formed by transesterification of cocaine in the presence of ethanol. Here we relate findings from a randomized, placebo-controlled, double-blind study in which we examined the physiological and subjective effects and pharmacokinetics of i.v. administered cocaethylene in human volunteers using cocaine as a comparator. METHODS: Cocaine-dependent participants randomly received one study drug, cocaethylene (0.25 or 0.5 mg/kg), cocaine (0.25 or 0.5 mg/kg), or placebo, during each experimental session which occurred on separate days. RESULTS: Cocaethylene was less potent in elevating heart rate than equivalent doses of cocaine. Similar differences between cocaine and cocaethylene were found for subjective measures ("Cocaine High", "Rush", "Stimulated" and "Good Drug Effects"). All active drug conditions produced significant increases in systolic blood pressure relative to placebo, but no significant effect on diastolic blood pressure was observed. Cocaethylene demonstrated a slower clearance, larger volume of distribution and correspondingly longer elimination half-life than cocaine. CONCLUSION: The findings from this study confirm those of previous studies that show that cocaethylene has pharmacological properties in common with cocaine, but is less potent.

Neurobiological adaptations to psychostimulants and opiates as a basis of treatment development.

Abuse of illicit substances, in particular psychostimulants and opiates, is a worldwide public health issue. Chronic use of cocaine and amphetamine causes common neurobiological adaptations that may guide new treatment development. These include perturbations in dopamine and serotonin neurotransmission, leading to trials of antidepressants, and serotonin and dopamine augmentation strategies. The detection of cerebral perfusion abnormalities caused by psychostimulants has led to examination of antiplatelet and excitatory amino acid (EAA) antagonist therapies. Further, development of cocaine vaccines allows for testing of peripheral blockade approaches to cocaine addiction. New approaches to behavioral treatments for cocaine dependence are also reviewed. For opiate dependence, understanding of heroin's effects on mu and kappa opiate receptors has led to investigations of the partial mu agonist buprenorphine in opiate maintenance. Evidence for hyper-excitability of locus coeruleus (LC) noradrenergic neurons and EEA inputs to the LC guides trials of new alpha 2-adrenergic agonists and EEA antagonists to alleviate opiate withdrawal. Finally, clinical experience with withdrawal from methadone and LAAM has led to trials of antagonist-accelerated opiate withdrawal. Improved treatment of psychostimulant and opiate addiction is critically needed, and likely to have wide-reaching impact in health care and society.

Processing and intracellular sorting of anglerfish and rat preprosomatostatins in mammalian endocrine cells.

Rat preprosomatostatin (rPPSS) is processed to two distinct end products in a tissue-specific manner. The analogous end products in anglerfish are derived from separate precursors, anglerfish preprosomatostatins-1 and -2 (a(1)PPSS and a(II)PPSS). This report reviews experiments demonstrating that in mammalian cells, the cell of expression, not precursor structure, determines the processing fate of the preprosomatostatins. A fusion precursor of a(II)PPSS and rPPSS was expressed in mammalian cell lines to determine that the amino-terminal 78 residues of rPPSS contain a sorting signal that directs the precursor into a regulated secretory pathway wherein proteolytic processing occurs. Preliminary studies of rPPSS pro-region mutations are presented that attempt to further localize this sorting signal.

Preprothyrotropin-releasing hormone mRNA in the rat central gray is strongly and persistently induced during morphine withdrawal.

In vivo evidence strongly implicates the central gray in expression of the physical symptoms of opiate withdrawal. Preprothyrotropin-releasing hormone (ppTRH) mRNA is highly expressed in the central gray. Furthermore, systemic administration of thyrotropin-releasing hormone (TRH) inhibits the development of opiate dependence in rats. To elucidate the link between TRH and opiate withdrawal, we examined the regulation of ppTRH mRNA in the central gray of rats made dependent on morphine, and during opiate withdrawal, using quantitative in situ hybridization. In the ventrolateral central gray, a significant increase in ppTRH mRNA was observed 3 h after precipitation of withdrawal, and this increase persisted for 36 h. Upregulation of ppTRH mRNA was not seen with chronic morphine or acute naltrexone treatment alone and was specific for the ventrolateral central gray. These findings support a role for TRH or other ppTRH-derived peptides in the central gray during morphine withdrawal.

A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function.

Previous studies have shown that chronic morphine increases levels of the G-protein subunits Gia and Goa, adenylate cyclase, cyclic AMP-dependent protein kinase, and certain phosphoproteins in the rat locus coeruleus, but not in several other brain regions studied, and that chronic morphine decreases levels of Gia and increases levels of adenylate cyclase in dorsal root ganglion/spinal cord (DRG-SC) co-cultures. These findings led us to survey the effects of chronic morphine on the G-protein/cyclic AMP system in a large number of brain regions to determine how widespread such regulation might be. We found that while most regions showed no regulation in response to chronic morphine, nucleus accumbens (NAc) and amygdala did show increases in adenylate cyclase and cyclic AMP-dependent protein kinase activity, and thalamus showed an increase in cyclic AMP-dependent protein kinase activity only. An increase in cyclic AMP-dependent protein kinase activity was also observed in DRG-SC co-cultures. Morphine regulation of G-proteins was variable, with decreased levels of Gia seen in the NAc, increased levels of Gia and Goa in amygdala, and no change in thalamus or the other brain regions studied. Interestingly, chronic treatment of rats with cocaine, but not with several non-abused drugs, produced similar changes compared to morphine in G-proteins, adenylate cyclase, and cyclic AMP-dependent protein kinase in the NAc, but not in the other brain regions studied. These results indicate that regulation of the G-protein/cyclic AMP system represents a mechanism by which a number of opiate-sensitive neurons adapt to chronic morphine and thereby develop aspects of opiate tolerance and/or dependence. The findings that chronic morphine and cocaine produce similar adaptations in the NAc, a brain region important for the reinforcing actions of many types of abused substances, suggest further that common mechanisms may underlie psychological aspects of drug addiction mediated by this brain region.

Cocaine regulation of brain preprothyrotropin-releasing hormone mRNA.

Clinical and preclinical evidence supports a possible role for thyrotropin-releasing hormone (TRH) in cocaine action. However, the interaction between cocaine and TRH has not been directly examined. In the following report we describe a solution hybridization RNase protection assay that can sensitively detect mRNA for the TRH precursor, prepro-TRH (ppTRH). Using this assay, we examined ppTRH mRNA levels in rat brain regions implicated in cocaine reinforcement, including the nucleus accumbens, hypothalamus, amygdala, hippocampus, and thalamus. Acute cocaine treatment (15 mg/kg) resulted in significant decreases in ppTRH mRNA levels in the amygdala and hippocampus, but not in the hypothalamus, nucleus accumbens, or thalamus, 45 min postinjection. Chronic cocaine treatment (15 mg/kg twice daily for 14 days) resulted in marked regulation in all regions but the thalamus. Regulation was strongly dependent on the length of cocaine withdrawal and persisted up to 72 h postinjection in the amygdala. These studies support the hypothesis that TRH or other ppTRH-derived peptides are involved in cocaine action, especially in the extrahypothalamic regions of the amygdala and hippocampus.

Mitochondrial membrane biogenesis: identification of a precursor to yeast cytochrome c oxidase subunit II, an integral polypeptide.

Many of the polypeptides made on endogenous ribosomes inside of yeast mitochondria are hydrophobic "integral polypeptides" which are subunits of at least three oligomeric enzyme complexes (cytochrome c oxidase, rutamycin-sensitive ATPase, and coenzyme QH2-cytochrome c reductase) of the inner mitochondrial membrane. In order to elucidate the pathway(s) followed by these polypeptides into the inner membrane we have used an in vitro mitochondrial translation system from yeast. By inhibiting this system with aurintricarboxylic acid, we have been able to demonstrate and accumulate a transient precursor to subunit II of cytochrome c oxidase. This precursor, designated II', is approximately 1,500 daltons larger than mature subunit II and most likely is a form of subunit II with an NH2-terminal extension. Although this precursor appears to be processed cotranslationally under normal conditions, it does associate in unprocessed form with mitochondrial membranes when allowed to accumulate in the presence of aurintricarboxylic acid, and it can be processed postranslationally upon removal of the drug. None of the other mitochondrial translation products made in this system exhibits larger precursors. These results indicate that at least one mitochondrial translation product has a transient "leader sequence" a,d is inserted into the inner mitochondrial membrane and processed cotranslationally, but they suggest that other pathways may be followed by the other translation products.

Regulation of cytochrome c oxidase subunit mRNA and enzyme activity in rat brain reward regions during withdrawal from chronic cocaine.

A subtractive hybridization and differential screening procedure was used to detect up-regulation of cytochrome c oxidase (CO) subunits I, III, and IV mRNA in the nucleus accumbens (NAc) of rats chronically treated with cocaine. Northern blot analyses of mRNA isolated from individual rats confirmed that CO subunit I was up-regulated by chronic, but not acute, cocaine in two brain regions, the NAc (33%) and caudate-putamen (CP) (35%). CO activity, used as a measure of metabolic activity, was increased by 88% in the NAc, and decreased by 20% in the medial prefrontal cortex (mPFC), the day after chronic treatment was terminated. CO enzyme activity was not regulated in the CP, or in other brain regions not involved in drug reward. CO activity in both the NAc and mPFC showed unique time-dependent patterns of regulation during the week after chronic cocaine treatment.

Multiple preprosomatostatin sorting signals mediate secretion via discrete cAMP- and tetradecanoylphorbolacetate-responsive pathways.

We have previously detected a sorting signal in the amino-terminal 78 residues of rat preprosomatostatin (rPPSS) that targets the precursor into a regulated secretory pathway or pathways allowing proteolytic maturation (Sevarino, K. A., Stork, P., Ventimiglia, R., Mandel, G., and Goodman, R. H. (1989) Cell 57, 11-19). To further localize this signal, we constructed three rPPSS expression vectors that code for substitutions or mutations spanning that portion of rPPSS implicated in sorting, and the precursors were expressed in RIN 5F cells. Fractionation of the intracellular products revealed that accurate processing to somatostatin-14 (SS-14) was not affected by any of the mutations. Examination of the secreted products showed no reduction in processing efficiency, indicating that none of the mutations blocked sorting from constitutive into regulated secretion. Finally, we examined the response to two separate secretogogues, cAMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Clones expressing two of the three mutant precursors displayed the same stimulation of SS-14 secretion by exogenously administered cAMP and TPA as cells expressing wild-type rPPSS, indicating that targeting specifically to the secretory pathway, or pathways, responsive to cAMP and TPA was not disrupted. However, cells expressing the mutant precursor containing a substitution of the amino-terminal 34 residues of rPPSS by the amino terminus of the vesicular stomatitis virus G protein displayed greatly reduced stimulation of SS-14 secretion by TPA, with a less than compensatory increase in response to cAMP, when compared to cells expressing wild-type rPPSS. In conjunction with our previous studies with anglerfish preprosomatostatins, we conclude that 1) the sorting signal(s) in rPPSS necessary for cAMP-responsive secretion are redundant and probably reside within both mature peptide regions and extrapeptide regions; 2) two or more distinct regulated secretory pathways utilized by secreted peptides can be demonstrated in transfected endocrine cells and targeting to these pathways can be separately mediated by at least two different types of sorting signals within the neuropeptide precursor itself; and 3) pro-region conformation plays little role in prosomatostatin-processing site recognition.

Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.

We have examined the regulation of somatostatin gene expression by cAMP in PC12 rat pheochromocytoma cells transfected with the rat somatostatin gene. Forskolin at 10 microM caused a 4-fold increase in somatostatin mRNA levels within 4 hr of treatment in stably transfected cells. Chimeric genes containing the somatostatin gene promoter fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase were also induced by cAMP in PC12 cells. To delineate the sequences required for response to cAMP, we constructed a series of promoter deletion mutants. Our studies defined a region between 60 and 29 base pairs upstream from the transcriptional initiation site that conferred cAMP responsiveness when placed adjacent to the simian virus 40 promoter. Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP. cAMP responsiveness was greatly reduced when the somatostatin fusion genes were transfected into the mutant PC12 line A126-1B2, which is deficient in cAMP-dependent protein kinase 2. Our studies indicate that transcriptional regulation of the somatostatin gene by cAMP requires protein kinase 2 activity and may depend upon a highly conserved promoter element.

Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity.

Rat preprosomatostatin (rPPSS) is processed to two bioactive peptides, somatostatin-14 and somatostatin-28. In anglerfish islets, the two peptides are synthesized by distinct cell types and are derived from different precursors, anglerfish preprosomatostatin-1 (a(I)PPSS) and anglerfish preprosomatostatin-2 (a(II)PPSS). To determine the basis of the differential processing, we introduced a(I)PPSS or a(II)PPSS expression vectors into mammalian endocrine cell lines that can accomplish both patterns of processing. Both precursors were processed identically, indicating that cellular factors must determine the processing pattern. Although similar processing sites are present in both precursors, high levels of unprocessed anglerfish prosomatostatin-2 were secreted constitutively from the transfected cells. A hybrid protein containing the leader sequence and a portion of the pro-region of rPPSS fused to the carboxy-terminal third of a(II)PPSS was processed and secreted via a regulated pathway. We conclude that the amino-terminal 78 residues of rPPSS contain sufficient information to correct the targeting deficiency of a(II)PPSS in mammalian endocrine cell lines.

Thyrotropin-releasing hormone (TRH) precursor processing. Characterization of mature TRH and non-TRH peptides synthesized by transfected mammalian cells.

Prepro-thyrotropin-releasing hormone (TRH) contains five TRH progenitor sequences and at least six other potential peptides (Lechan, R. M., Wu, P., Jackson, I. M. D., Wolf, H., Cooperman, S., Mandel, G., and Goodman, R. H. (1986a) Science 231, 159-161). Previous studies using radioimmunoassays developed against discrete regions of prepro-TRH have demonstrated that several of the potential peptides are present in rat brain and pancreas (Wu, P., Lechan, R. M., and Jackson, I. M. D. (1987) Endocrinology 121, 108-115; Wu, P. and Jackson, I. M. D. (1988a) Brain Res. 456, 22-28; Wu, P., and Jackson, I. M. D. (1988b) Regul. Pept. 22, 347-360). However, the low level of peptides present in intact tissues has made isolation of the peptides difficult. CA77 cells, a medullary thyroid carcinoma cell line, also express prepro-TRH and display processing similar to that found in tissues. However, peptide content in this tumor cell line is enhanced only 3-fold compared with normal tissues (Sevarino, K. A., Wu, P., Jackson, I. M. D., Roos, B. A., Mandel, G., and Goodman, R. H. (1988) J. Biol. Chem. 263, 620-623). To achieve higher levels of expression for facilitating peptide sequencing studies and to see if alternate processing of prepro-TRH could be detected in different cell types, we transfected into 3T3, GH4, AtT20, and RIN 5F cells a cDNA vector under control of the cytomegalovirus immediate-early promoter. 3T3 and GH4 cells failed to process prepro-TRH beyond cleavage of the signal sequence. Both AtT20 and RIN 5F cells efficiently cleaved the precursor at dibasic sites to generate mature TRH and the non-TRH peptides previously identified in vivo. Peptide content was up to 30 times greater than in hypothalamic extracts and 10 times greater than in CA77 cells. Secretion experiments with transfected AtT20 cells demonstrated that both mature TRH and the non-TRH peptides were secreted via a regulated secretory pathway similar to that utilized by endogenously synthesized peptides. We isolated several of the non-TRH peptides synthesized by transfected AtT20 cells and characterized these peptides by sequential Edman degradation. These studies identified the signal sequence cleavage site and determined that the non-TRH peptides are generated by cleavage at the dibasic sites flanking the five TRH progenitor sequences. Further, we determined that processing occurs at the Arg51-Arg52 site located in the amino-terminal portion of the precursor, the only dibasic site not flanking a TRH progenitor sequence.

Cloning and characterization of chi-1: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family.

Ionotropic glutamate receptors are composed of homomeric or heteromeric configurations of glutamate receptor subunits. We have cloned a member of a novel class of the rat ionotropic glutamate receptor family, termed chi-1. This subunit exhibits an average identity of 27% to NMDA subunits and 23% to non-NMDA subunits. Regional transcript levels of chi-1 are elevated just prior to and during the first postnatal week, with the highest levels present in the spinal cord, brainstem, hypothalamus, thalamus, CA1 field of the hippocampus, and amygdala. The spatial distribution of chi-1 expression is similar from postnatal day 1 (P1) to adulthood. However, transcript levels decline sharply between P7 and P14 and remain attenuated into adulthood. Functional expression studies in Xenopus oocytes injected with in vitro transcribed chi-1 RNA did not demonstrate agonist-activated currents. Pairwise expression of chi-1 with members of the AMPA, KA, or delta class of glutamate recepto subunits either failed to generate agonist-activated currents or failed to alter the underlying current generated by the coexpressed subunit. However, coexpression of chi-1 with subunits forming otherwise functional NMDA receptors resulted in an inhibition of current responses. Since chi-1 did not alter the currents generated by non-NMDA subunits, this suggests that chi-1 may specifically interact with NMDA receptor subunits. Further characterization will be required to establish the precise role of this glutamate receptor subunit in neuronal signaling.

Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line.

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.