Blockade Of Protein Synthesis Research Articles

Anatomical studies of DNA fragmentation in rat brain after systemic kainate administration

Rats treated systemically with kainate develop stereotyped epileptic seizures involving mainly limbic structures that may last for hours. This model of limbic status epilepticus has been widely studied using classical neuropathological techniques. We used in situ nick translation histochemistry to examine patterns of DNA fragmentation in this model. We found a stereotyped and reproducible pattern of neuronal populations that demonstrate evidence of DNA fragmentation from 24 h to one week after kainate treatment. Neither blockade of new protein synthesis nor blockade of the N-methyl- d-aspartate-type glutamate receptors significantly altered this response. Moreover, we saw no evidence of the regular internucleosomal cleavage of DNA that produces a characteristic laddered appearance of 180–200 bp DNA fragments after gel electrophoresis in samples obtained from microdissected affected regions. These studies suggest that DNA fragmentation after systemic kainate-induced seizures is not the result of programmed cell death. This assay may be useful for quantitative testing of both neuroprotective agents and mechanistic hypotheses.

Evidence That Syntaxin 1A Is Involved in Storage in the Secretory Pathway

Syntaxin 1A is a nervous system-specific protein thought to function during the late steps of the regulated secretory pathway by mediating the docking of secretory vesicles with the plasma membrane. We have examined the effects of transiently overexpressing syntaxin 1A on protein secretion in constitutively secreting cell lines that do not normally express the protein. Syntaxin 1A showed the constitutive release of marker proteins human growth hormone (hGH) and vesicular stomatitis virus glycoprotein from COS-1 cells, increasing the intracellular half-life of human growth hormone from 90 min to 18 h. A similar effect was observed in HEK 293 cells. Immunofluorescence microscopy revealed that these secretory proteins were concentrated in the periphery of the cell. The effect was specific for the full-length neuronal protein. Neither a syntaxin 1A variant which lacks a membrane attachment domain nor syntaxin 2 caused the cells to retain human growth hormone. The effect of syntaxin 1A was partially reversed by incubating the cells with botulinum type C1 neurotoxin, which specifically cleaves syntaxin 1A. Release of human growth hormone from syntaxin 1A-expressing cells was maintained during a blockade of protein synthesis, suggesting that the hormone was being released from a pool of stored vesicles which accumulated before the addition of cycloheximide. The existence of a post-Golgi storage compartment in syntaxin 1A-expressing cells was confirmed using brefeldin A to collapse the Golgi stacks in both HEK 293 and COS-1 cells. Brefeldin A rapidly blocked growth hormone release in control cultures while having no effect on release in cells expressing syntaxin 1A. Reducing the temperature to 19 degrees C, which inhibits transport from the trans-Golgi network, also inhibited hGH secretion from cells without syntaxin 1A but had little effect on hGH secretion from cells with syntaxin 1A. The present experiments indicate that syntaxin 1A enables the storage of vesicles which would otherwise be immediately released.

Morphological plasticity in dendritic spines of cultured hippocampal neurons.

Rat hippocampal neurons, grown in dissociated culture for about 18 days, were exposed for 6 h to three days to stimuli which cause either an increase (GABAA antagonists, bicuculline or picrotoxin), or decrease (tetrodotoxin) in spontaneous neuronal activity. Individual neurons were stained with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate and visualized with a confocal laser scanning microscope. GABA antagonists caused a marked, up to 60%, increase in spine density on secondary dendrites of cultured hippocampal neurons. This was associated with a small decrease in spine length. The rise in spine density was partially prevented by treatment with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetra-acetate, or by blockade of protein synthesis with cycloheximide. Tetrodotoxin caused a marked elongation of dendritic spines (but did not cause a decrease in spine density comparable to the increase caused by picrotoxin). This effect was seen primarily but not exclusively in spines with no distinct head. Both treatments were most effective within 24 h of exposure. There were no other systematic effects of the drugs on the morphology of the dendritic spines. These results indicate that dendritic spines in cultured neurons depend on ongoing synaptic activity to maintain their shape, and that neurons respond to an increase in synaptic demand by an increase in spine density. Thus, dendritic spines are likely to have a role in short-term synaptic interaction rather than to constitute a long-term memory storage device.

Post-translational regulation of neuronal acetylcholine receptors stably expressed in a mouse fibroblast cell line.

Second messenger regulation of neuronal acetylcholine receptors (AChRs) was investigated in a mouse fibroblast cell line, M10, stably transfected with chicken alpha 4 and beta 2 cDNAs. Both forskolin and 8-bromo-cyclic adenosine 3',5'-monophosphate (cAMP) induced large increases in the numbers of AChRs. The increases were due in part to increased transcription and translation of the alpha 4 and beta 2 genes. Blockade of protein synthesis with cycloheximide, however, revealed that forskolin also exerts a post-translational effect, increasing the number of surface receptors by twofold. Immunoblot analysis of sucrose gradient fractions confirmed that the cells had a large fraction of unassembled subunits potentially available for receptor assembly. The post-translational effect of forskolin was blocked by H-89, an inhibitor of cAMP-dependent protein kinase, and by okadaic acid, an inhibitor of phosphatases 1 and 2A. Nicotine also acted post-translationally to induce a twofold increase in the number of surface receptors, but the mechanism differed from that utilized by forskolin, since the effects of the two agents were additive and were differentially affected by okadaic acid. The results suggest that protein phosphorylation-dephosphorylation mechanisms act post-translationally to increase the number of neuronal AChRs maintained on the cell surface. This could be achieved by increasing the efficiency of receptor assembly, transport, or stabilization on the cell surface.

Cyclic AMP-independent Up-regulation of the Human Serotonin Transporter by Staurosporine in Choriocarcinoma Cells

Treatment of confluent cultures of JAR human placental choriocarcinoma cells with staurosporine caused a marked stimulation of serotonin transport activity in these cells. The stimulatory effect was noticeable at nanomolar concentrations of staurosporine, and a treatment time of > 4 h was required for staurosporine to elicit the effect. At 40 nM and with a treatment time of 16 h, the stimulation of the transport activity was 3.5-6.0-fold. None of the several other protein kinase inhibitors tested had similar effect except KT 5720, a protein kinase A inhibitor, which showed a small but significant (approximately 1.4-fold) stimulatory effect at a concentration of 5 microM. Blockade of RNA synthesis and protein synthesis in the cells prevented completely the stimulation of the transport activity induced by staurosporine. The stimulation was observed not only in intact cells but also in plasma membrane vesicles prepared from staurosporine-treated cells. The stimulation was accompanied by a 5-7-fold increase in the steady state levels of the transporter-specific mRNAs, by a 7-fold increase in the maximal velocity of the transport process, and by a 6-fold increase in the transporter density in the plasma membrane. Even though both staurosporine and cholera toxin had similar effects on the serotonin transport activity in these cells, the effect was not additive when the cells were treated with both reagents together. While treatment of the cells with cholera toxin markedly elevated intracellular levels of cAMP, staurosporine did not have any effect on the cellular levels of this cyclic nucleotide. It is concluded that staurosporine up-regulates the serotonin transport activity in JAR cells by increasing the steady state levels of the serotonin transporter mRNA and by the consequent increase in the transporter density in the plasma membrane and that the process involves a cAMP-independent signaling pathway.

Rapid activation of the stat3 transcription complex in liver regeneration

Liver regeneration in response to partial hepatectomy is a physiological growth response observed in the intact animal. Understanding the early signals that trigger liver regeneration is of vital importance to understand the liver's response to injury. It has been observed that several growth factors and cytokines, including epidermal growth factor (EGF) and interleukin-6 (IL-6), can activate members of the signal transducers and activators of transcription (Stat) family of transcription factors resulting in tyrosine phosphorylation of these factors, nuclear translocation, and an active DNA binding transcriptional complex. Because Stat3 participates in the regulation of primary growth response genes, we wondered if it is induced in the early phase of liver regeneration. We found that Stat3 DNA-binding activity is increased in the remnant liver within 30 minutes of partial hepatectomy and peaks at more than 30-fold at 3 hours. This induction is not observed after sham surgery. The induction of Stat3 appears to be part of the initial response of the remnant liver to partial hepatectomy, because it occurs in the presence of cycloheximide-mediated protein synthesis blockade. Activation of Stat3 is unusual, because it extends beyond the immediate-early time period and remains near peak level at 5 hours posthepatectomy. Although insulin-treated H35 cells activate many of the same immediate-early genes as regenerating liver, Stat3 is not induced in these cells. Because Stat factors are known to be inactivated by protein tyrosine phosphatases (PTPase), we showed that a PTPase is able to eliminate the DNA binding of hepatic Stat3.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid activation of the Stat3 transcription complex in liver regeneration

Liver regeneration in response to partial hepatectomy is a physiological growth response observed in the intact animal. Understanding the early signals that trigger liver regeneration is of vital importance to understand the liver's response to injury. It has been observed that several growth factors and cytokines, including epidermal growth factor (EGF) and interleukin-6 (IL-6), can activate members of the signal transducers and activators of transcription (Stat) family of transcription factors resulting in tyrosine phosphorylation of these factors, nuclear translocation, and an active DNA binding transcriptional complex. Because Stat3 participates in the regulation of primary growth response genes, we wondered if it is induced in the early phase of liver regeneration. We found that Stat3 DNA-binding activity is increased in the remnant liver within 30 minutes of partial hepatectomy and peaks at more than 30-fold at 3 hours. This induction is not observed after sham surgery. The induction of Stat3 appears to be part of the initial response of the remnant liver to partial hepatectomy, because it occurs in the presence of cycloheximide-mediated protein synthesis blockade. Activation of Stat3 is unusual, because it extends beyond the immediate-early time period and remains near peak level at 5 hours posthepatectomy. Although insulin-treated H35 cells activate many of the same immediate-early genes as regenerating liver, Stat3 is not induced in these cells. Because Stat factors are known to be inactivated by protein tyrosine phosphatases (PTPase), we showed that a PTPase is able to eliminate the DNA binding of hepatic Stat3. It is likely that Stat3 contributes to the transcriptional activation of a subset of immediate-early genes that are induced over a prolonged time in the G1 phase of hepatic cells following partial hepatectomy. The identification of Stat3 as an early factor in liver regeneration provides clues to the activation of signal transduction pathways in the remnant liver within the first minutes to several hours after partial hepatectomy.

Activity-dependent regulation of a ribosomal RNA epitope in the chick cochlear nucleus

Elimination of auditory nerve activity results in rapid metabolic changes, cell atrophy, and cell death in nucleus magnocellularis (NM), the cochlear nucleus of the chick. The transneuronal signals involved in the activity-dependent regulation of NM neurons are not well understood. One of the most rapid transneuronal effects is alteration in protein synthesis by NM neurons. Previous studies using an in vitro preparation of the brain stem auditory system suggested that up-regulation of protein synthesis in NM neurons requires the action of some trophic substance released by active auditory nerve fibers. Here, similar results were obtained when measuring changes in immunoreactivity using a monoclonal antibody (Y10B) that recognizes ribosomal RNA. This immunolabeling assay has advantages over the global protein synthesis assay in that it is not sensitive to possible changes in specific activity of the precursor pool or possible differences in the uptake of the labeled amino acids. Unilateral stimulation of the auditory nerve for 1 h resulted in greater immunolabeling of NM neurons on the stimulated side of the slice. This is consistent with previous in vivo results after unilateral deafferentation. Blockade of synaptic transmission by maintaining the slice in a low-Ca 2+/high Mg 2+ medium prevented the stimulation-induced difference in immunolabeling. Electrical stimulation of the postsynaptic NM neurons alone (antidromic stimulation, via electrical stimulation of NM neuron axons) did not result in greater immunolabeling. Rather, antidromically stimulated neurons tended to show lighter labeling. Thus, the transneuronal regulation of ribosomes in NM neurons appears to require some substance released from the active auditory nerve. Further, blockade of protein synthesis using cycloheximide did not prevent the activity-dependent difference in immunolabeling. Thus, the activity-dependent regulation of ribosomes does not appear to require new protein synthesis during the period of differential activity.

Increased deafferentation-induced cell death in chick brainstem auditory neurons following blockade of mitochondrial protein synthesis with chloramphenicol

Second-order auditory neurons in nucleus magnocellularis (NM) of the chick brainstem undergo a series of rapid metabolic changes following unilateral cochlea removal, culminating in the death of 25% of NM neurons. Within hours of cochlea removal, ipsilateral NM neurons show marked increases in histochemical staining for the mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase (CO). We have shown previously in an ultrastructural study that these increases in oxidative capacity are mediated in part by a rapid increase in mitochondrial volume within deafferented neurons. In neurons that are destined to die as a result of deafferentation, mitochondria are smaller, stain poorly for CO, and often contain vacuoles associated with oxidative dysfunction. Our present set of experiments is designed to test the hypothesis that increases in oxidative metabolism are necessary for NM neuronal survival following removal of afferent input. We used chloramphenicol (CAP), a mitochondrial protein synthesis inhibitor, to block the characteristic increase in CO activity and mitochondrial proliferation following cochlea removal. We then studied the effects of CAP on NM neuronal survival following deafferentation. When CAP was administered continuously at 600 mg/kg/d for 5 d following cochlea removal, deafferentation-induced neuronal death in NM was significantly increased from 22% to 36%. Higher-dose (1200 mg/kg/d) pulses of CAP were administered for the first 6, 12, or 24 hr following cochlea removal. After 5 d survival, greater increases in neuronal cell death were found in animals treated for the first 12 or 24 hr (65% neuronal death). CAP administration for the first 6 hr had no significant effect on neuronal survival. The effects of CAP on neuronal cell death in NM are not likely to be due to systemic effects of the drug, but instead to a specific change in mitochondrial function. Our results suggest that enhanced oxidative enzyme function plays an important role in the survival of NM neurons during the first 24 hr after deafferentation. The nature of the signal(s) eliciting mitochondrial enhancement and the means by which it influences NM survival are not known.

Calcitonin stimulates calcium transport in distal convoluted tubule cells

Calcitonin increases Ca2+ absorption by renal distal tubules. The mechanism underlying this action is not known. Calcitonin effects on Ca2+ uptake, intracellular Ca2+ activity, and membrane voltage were determined in transformed mouse distal convoluted tubule (DCT) cells. Intracellular Ca2+, measured in single cells grown on cover slips and loaded with fura-2, averaged 102 nM. Maximal increases of intracellular Ca2+ to 312 nM were elicited by 1 nM calcitonin. Calcitonin stimulated 45Ca2+ influx by 45% from control rates of 2.8 nmol.min-1 x mg protein-1. Membrane voltage was measured in single cells loaded with the voltage-sensitive dye 3,3'-dihexyloxacarbocyanine iodide. Resting membrane voltage averaged 69 mV. Calcitonin maximally hyperpolarized cells by 18 mV. The Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid, abolished calcitonin-induced membrane hyperpolarization and Ca2+ influx. Calcitonin increased halide ion permeability, estimated from the rate of 125I- efflux, by 160%. The Ca2+ channel blocker nifedipine abolished calcitonin-stimulated 45Ca2+ uptake and the increase of intracellular Ca2+. Combined exposure to calcitonin and parathyroid hormone (PTH) had no additional effect on 45Ca2+ influx or intracellular Ca2+. In contrast to the long latency (8 min) before onset of PTH effects, calcitonin increased 45Ca2+ uptake and intracellular Ca2+ concentration within 2 min. Blockade of protein synthesis with cycloheximide inhibited Ca2+ uptake induced by PTH but not that stimulated by calcitonin. We conclude that calcitonin increases Cl- conductance in DCT cells, resulting in membrane hyperpolarization and activation of Ca2+ entry through dihydropyridine-sensitive Ca2+ channels. The shorter onset of calcitonin action compared with PTH may reflect differences in the signaling mechanisms between the two hormones.

Timing of neuronal death following successive blockade of protein synthesis and axoplasmic transport in the axonal target territory.

Neurons in the isthmo-optic nucleus (ION) of chick embryos can withstand a substantial cycloheximide-induced reduction in protein synthesis in their target territory, the retina, at the very time when their survival is known to depend on a retrograde signal from the latter. We here test the hypothesis that this resistance to the cycloheximide injection might be due to the accumulation in the ION of a reserve of retina-derived trophic molecules (or of resulting second messengers). Following an intraocular injection of cycloheximide at E15 to deplete the hypothetical reserve in one ION, both eyes received injections of colchicine (which blocks axoplasmic transport) at E16. The resulting time course of cell death was very similar in the two IONs, which refutes the hypothesis. The most plausible alternative is that there is a reserve of trophic substance in the retina capable of maintaining the ION for about 1 day.

Pulse exposure to protein synthesis inhibitors enhances vascular responses to des‐Arg9‐bradykinin: possible role of interleukin‐1

1. The modulation of the spontaneous increase in contractile responses to des-Arg9-bradykinin (des-Arg9-BK) of rabbit aortic strips incubated in vitro was studied. Rapid hypotensive responses to exogenous kinins were also measured in rabbits anaesthetized 5 h following pretreatment. 2. Continuous exposure to the protein synthesis inhibitors cycloheximide (71 microM) or anisomycin (3.8 microM) profoundly inhibited the sensitization to des-Arg9-BK in incubated aortic strips. However, temporary (3 h) inhibition of protein synthesis in vitro followed by further incubation (3 h) of tissues without inhibitor, paradoxically enhanced both the maximal contractile responses to des-Arg9-BK (1.7 microM) and the apparent affinity of the kinin without affecting contractions to noradrenaline (NA, 100 nM) at 6.5 h. 3. The stimulatory activity of the short treatment (pulse) with cycloheximide was abolished in the presence of dexamethasone sodium phosphate (100 microM throughout the incubation). The function of receptors for kinins did not appear to be altered directly by the steroid treatment. 4. Interleukin-1 beta (IL-1 beta), applied at low concentrations (100-250 pg ml-1) on aortic strips between 3 h and 6.5 h of incubation time, mimicked the selective stimulatory effect of the cycloheximide pulse on responses to des-Arg9-BK. Higher concentrations of IL-1 beta (0.5-5 ng ml-1) did not further amplify the responses to des-Arg9-BK but decreased the contractile responses to NA. 5. The modulation by IL-1 beta of vascular sensitivity to des-Arg9-BK and to NA was prevented by blockade of protein synthesis. 6. The induction in vivo by IL-1 beta (5 micrograms kg-1) or by cycloheximide (10 mg kg-1) of cardiovascular responsiveness to des-Arg9-BK was demonstrated with a blood pressure assay of exogenous kinins or with tissues isolated ex vivo 5 h after pretreatment of animals. Evidence of active disposition of cycloheximide in vivo was also obtained. 7. We propose the production of endogenous IL-1 as a possible mechanism for the enhancement of responsiveness to des-Arg9-BK observed in tissues pulsed with a protein synthesis inhibitor and for the inducing effect of cycloheximide or E. coli lipopolysaccharide in vivo. These results suggest that effects mediated by the BK1 receptor for kinins are potentially present in pathological conditions associated with IL-1 production.

Effect of protein synthesis Inhibition on brain corticotropin-releasing factor and plasma adrenocorticotropin

The effect of inhibiting protein synthesis on concentrations of corticotropin-releasing factor (CRF) in rat brain and plasma adrenocorticotropin (ACTH) was assessed following the administration of the general protein synthesis inhibitor anisomycin. Compared with vehicle-injected controls, protein synthesis inhibition resulted in significantly reduced CRF immunoreactivity (CRF-ir) in median eminence within 1 h (p less than 0.01), remained decreased after 4 h (p less than 0.025), and was nonsignificantly decreased after 24 h. Plasma ACTH levels were greatly increased within 1 h posttreatment (p less than 0.0005), continued elevated after 4 h (p less than 0.01), and returned to normal levels after 24 h. CRF-ir measured in other brain areas 24 h after anisomycin showed decreased levels in medulla-pons (p less than 0.025) and neurointermediate lobe of pituitary (p less than 0.05), with no change noted in frontal cortex, hippocampus, midbrain-thalamus, or cerebellum. Overall these data show that blockade of normal protein synthesis with anisomycin can elicit changes in CRF-ir and ACTH content.

Effect of cycloheximide blockade of protein synthesis in rats on alcohol motivation

Effect of cycloheximide blockade of protein synthesis in rats on alcohol motivation

Electrophysiological and ultrastructural studies on reversible neural conduction disturbance after high voltage discharge

High-voltage condenser discharges exerting a field strength of up to 1000 V/cm (discharge time constant 0.24-8 msec) applied to isolated sciatic frog nerve lead to disturbances of the propagation of action potentials including transient complete block of conduction. Such conduction disturbances are normally reversible within minutes. Inhibition of the activity of the membrane-bound Na+-K+ATPase prevents the recovery from conduction block. Withdrawal of external Ca2+ also prevents recovery, whereas blockade of protein synthesis by cycloheximide has no influence. The velocity of recovery depends on the temperature, with temperature coefficients (Q10) from 1.31 to 1.84 between 2 degrees and 30 degrees C. Transmission electron microscopy of nerves subjected to strong discharges shows alterations of the myelin sheath (splitting and cleft formation) which are, however, not specific for this mechanism of injury. No alterations are seen in the region of the free axoplasmic membrane of the node of Ranvier or in organelles. The results suggest a breakdown of the transmembrane ionic gradient causing the conduction disturbance.

Modification of membrane permeability during semliki forest virus infection

Modification of membrane permeability has been analyzed in Semliki Forest virus (SFV)-infected cells by means of translation inhibitors not permeable to normal cells. A higher inhibition of protein synthesis in the infected cells is only observed with those antibiotics that do not easily pass the cell membrane, but not with others, permeable to cells, such as anisomycin, cycloheximide, trichodermin, etc. It does not, therefore, seem that the suggestion of M. A. Gray, K. J. Micklem, and C. A. Pasternak [ Eur. J. Biochem. 135, 299–302, (1983)] that protein synthesis in virus-infected cells is more susceptible to translation inhibitors in general, is correct. Both low- and high-molecular weight compounds enter the cell very early during SFV infection. This permeabilization is blocked by compounds known to increase the pH of coated vesicles, such as NH 4Cl and chloroquine. Inhibition of energy production by means of N 3Na and 2′-deoxyglucose also blocks this process. The optimal external pH for this early permeabilization is around 7–8. Acidic pH inhibits the entry of these impermeant antibiotics promoted by SFV. Analysis of 86Rb + content in SFV-infected HeLa cells also indicates that a drastic decline in this cation takes place, in agreement with previous findings, but disagreeing with the previous results. A parallel between the decrease in this cation and the blockade of protein synthesis is apparent, throughout the course of infection. In addition to the early permeabilization that takes place during virus entry, increased entry of hygromycin B and a-sarcin also occurs in SFV-infected cells from 2 to 3 hr postinfection, but not when late viral replication is blocked by means of interferon treatment.

The effect of serum deprivation on the initiation of protein synthesis in mouse neuroblastoma cells

Growth of mouse neuroblastoma cells becomes stationary when cultured in serum-free medium. Within 60 h, the protein-synthesizing capacity of the cells declines to 25% as compared to that of exponentially growing cells. The translational activity of the crude ribosomal salt washes from serum-deprived and control cells was compared in in vitro protein-synthesizing pH 5 systems. It appears that the ribosomal salt wash from serum-deprived cells has significantly (70%) lost its ability to support the translation of neuroblastoma poly(A) + RNA. This activity of the ribosomal wash from serum-deprived cells can be restored to control level with rabbit reticulocyte initiation factor eIF-4B only. The ability of the ribosomal wash from serum-deprived cells to support the translation of encephalomyocarditis virus (EMC) and Semliki Forest virus (SFV) 42 S mRNA was tested. We found that EMC-mRNA is efficiently translated with the ribosomal salt wash from serum-deprived cells, whereas on the other hand the translation of SFV 42 S mRNA is severely impaired. Therefore, we conclude that in serum-deprived neuroblastoma cells protein synthesis is regulated in both a quantitative and a qualitative way. Modulation of the activity of initiation factor of protein synthesis eIF-4B is at least partly responsible for the observed (selective) blockade of protein synthesis in serum-deprived cells.

Development and regulation of substance P in sensory neurons in vitro

Substance P (SP), the putative neuropeptide mediator of pain sensation, is contained in small dorsomedial sensory neurons of the dorsal root ganglion. Using different culture techniques and a sensitive radioimmunoassay for SP, we studied the ontogeny and regulation of this functionally important neurotransmitter in these neurons, obtained from neonatal rats. In ganglion explants grown by two different techniques, SP increased two- to threefold during the first week in culture. This rise was predominantly due to mechanisms intrinsic to the ganglion since it occurred in a fully defined medium, in the absence of added nerve growth factor (NGF). Blockade of protein synthesis with cycloheximide prevented the increase in SP suggesting that ongoing protein synthesis was necessary. Furthermore, depolarization with veratridine blocked the increase in SP, an effect which was reversed by tetrodotoxin, suggesting that transmitter characteristics in sensory neurons may be regulated by depolarization and/or transmembrane sodium flux. After a week in culture on a collagen substratum, supplementary NGF was necessary for the continued rise in SP. However, raising the dose of the trophic factor had no incremental effect on SP content, suggesting that NGF was acting primarily on neuronal survival. To approach such questions at the cellular level, ganglia were dissociated and grown in cell culture. In all cultures, SP increased 1.5-fold during the first day. In the absence of NGF, however, SP and cell numbers fell progressively after the second day. NGF elicited parallel increases in cell survival and SP content, supporting the suggestion that NGF acts primarily through neuronal survival to increase SP. Veratridine blocked the increase in SP in a tetrodotoxin-reversible manner, without affecting neuronal survival, indicating that the effects of these agents do not depend on normal ganglionic cellular architecture. Consequently, depolarization probably affects ganglionic sensory neurons directly. Our studies suggest that the development of transmitter characteristics in primary sensory neurons may be regulated by multiple factors, including neuronal activity as well as trophic agents such as NGF.

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection.

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-alpha (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon-treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.

Independence of beta-adrenergic stimulation of renin release on renin synthesis.

Rat renal cortical slices incubated in vitro released 7.0 +/- 0.5% (mean +/- SE, n=30) of their total renin content into the incubation medium in 1 h. Isoproterenol (10-6 M), a beta-adrenergic agonist, caused a 75 +/- 17% (n=7) increase in renin release from the cortical slices. Treatment of the cortical slices with either cycloheximide or puromycin caused a significant 96.2 +/- 0.34 (n=8) or 98.5 +/- 0.3% (n=5), respectively, inhibition of protein synthesis. After protein synthesis and presumably renin synthesis was blocked with either cycloheximide or puromycin, isoproterenol was still able to increase significantly renin release from the cortical slices despite almost total blockade of protein synthesis. We conclude that a storage pool of renin content is released may exist, since 1) only 7.0 +/- 0.5% of the cortical slice renin content is released each hour, and 2) isoproterenol stimulation of renin release is not acutely dependent on renin synthesis. It is hypothesized that beta-adrenergic stimulation of renin release is elicited from a storage pool of previously synthesized renin.