Rat vagus nerve stimulation model of seizure suppression: nNOS and ΔFos B changes in the brainstem.

Vagus nerve stimulation (VNS) is a moderately effective treatment for intractable epilepsy. However, the mechanism of action is poorly understood. The effect of left VNS in amygdala kindled rats was investigated by studying changes in nNOS and ΔFos B expression in primary and secondary vagus nerve projection nuclei: the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve (DMV), parabrachial nucleus (PBN) and locus coeruleus (LC). Rats were fully kindled by stimulation of the amygdala. Subsequently, when the fully kindled state was reached and then maintained for ten days, rats received a single 3-min train of VNS starting 1min prior to the kindling stimulus and lasting for 2min afterwards. In control animals the vagus nerve was not stimulated. Animals were sacrificed 48h later. The brainstems were stained for neuronal nitric oxide synthase (nNOS) and ΔFos B. VNS decreased seizure duration with more than 25% in 21% of rats. No VNS associated changes in nNOS immunoreactivity were observed in the NTS and no changes in ΔFos B were observed in the NTS, PBN, or LC. High nNOS immunopositive cell densities of >300cells/mm(2) were significantly more frequent in the left DMV than in the right (χ(2)(1)=26.2, p<0.01), independent of whether the vagus nerve was stimulated. We conclude that the observed nNOS immunoreactivity in the DMV suggests surgery-induced axonal damage. A 3-min train of VNS in fully kindled rats does not affect ΔFos B expression in primary and secondary projection nuclei of the vagus nerve.

Neural pathways associated with loss of consciousness caused by intracerebral microinjection of GABA A-active anesthetics.

Anesthesia, slow-wave sleep, syncope, concussion and reversible coma are behavioral states characterized by loss of consciousness, slow-wave cortical electroencephalogram, and motor and sensory suppression. We identified a focal area in the rat brainstem, the mesopontine tegmental anesthesia area (MPTA), at which microinjection of pentobarbital and other GABA(A) receptor (GABA(A)-R) agonists reversibly induced an anesthesia-like state. This effect was attenuated by local pre-treatment with the GABA(A)-R antagonist bicuculline. Using neuroanatomical tracing we identified four pathways ascending from the MPTA that are positioned to mediate electroencephalographic synchronization and loss of consciousness: (i) projections to the intralaminar thalamic nuclei that, in turn, project to the cortex; (ii) projections to several pontomesencephalic, diencephalic and basal forebrain nuclei that project cortically and are considered parts of an ascending "arousal system"; (iii) a projection to other parts of the subcortical forebrain, including the septal area, hypothalamus, zona incerta and striato-pallidal system, that may indirectly affect cortical arousal and hippocampal theta rhythm; and (iv) modest projections directly to the frontal cortex. Several of these areas have prominent reciprocal projections back to the MPTA, notably the zona incerta, lateral hypothalamus and frontal cortex. We hypothesize that barbiturate anesthetics and related agents microinjected into the MPTA enhance the inhibitory response of local GABA(A)-R-bearing neurons to endogenous GABA released at baseline during wakefulness. This modulates activity in one or more of the identified ascending neural pathways, ultimately leading to loss of consciousness.

Projections from the mesopontine tegmental anesthesia area to regions involved in pain modulation.

Pentobarbital microinjected into a restricted locus in the upper brainstem induces a general anesthesia-like state characterized by atonia, loss of consciousness, and pain suppression as assessed by loss of nocifensive response to noxious stimuli. This locus is the mesopontine tegmental anesthesia area (MPTA). Although anesthetic agents directly influence spinal cord nociceptive processing, antinociception during intracerebral microinjection indicates that they can also act supraspinally. Using neuroanatomical tracing methods we show that the MPTA has multiple descending projections to brainstem and spinal areas associated with pain modulation. Most prominent is a massive projection to the rostromedial medulla, a nodal region for descending pain modulation. Together with the periaqueductal gray (PAG), the MPTA is the major mesopontine input to this region. Less dense projections target the PAG, the locus coeruleus and pericoerulear areas, and dorsal and ventral reticular nuclei of the caudal medulla. The MPTA also has modest direct projections to the trigeminal nuclear complex and to superficial layers of the dorsal horn. Double anterograde and retrograde labeling at the light and electron microscopic levels shows that MPTA neurons with descending projections synapse directly on spinally projecting cells of rostromedial medulla. The prominence of the MPTA's projection to the rostromedial medulla suggests that, like the PAG, it may exert antinociceptive actions via this bulbospinal relay.

Porcine intrinsic cardiac ganglia.

The gross, light, and electron microscopic anatomies of the porcine intrinsic cardiac nervous system were investigated in 26 pigs to facilitate functional studies in this model. Gross anatomy: Numerous ganglia and interconnecting nerves (ganglionated plexuses) were found to be concentrated in epicardial fat in five atrial and six ventricular regions. The five atrial ganglionated plexuses identified were (1) the ventral right atrial, (2) the right vena cava-right atrial, (3) the dorsal atrial, (4) the interatrial septal, and (5) the left superior vena cava-left atrial ones. Six ventricular ganglionated plexuses were identified in close proximity to the (1) roots of the aorta and pulmonary artery (craniomedial), extending along the left main coronary artery to the (2) ventral interventricular and (3) circumflex coronary arteries. (4) A ganglionated plexus was identified around the origin of the dorsal interventricular coronary artery, as well as the (5) right main and (6) right marginal coronary arteries. Isolated neurons were identified scattered throughout the cranial interventricular septum. Microscopic anatomy: Approximately 3,000 neuronal somata were estimated to compose this intrinsic cardiac nervous system. Some ganglia contained more than 100 neurons. Neuronal somata had dimensions of roughly 33.1 (short axis) by 46.3 (long axis) microm. Most were multipolar, a small population of unipolar neurons being identified in atrial and ventricular tissues. At the electron microscopic level, asymmetrical axodendritic synapses with small clear, round vesicles were identified, some containing large dense-cored vesicles. In summary, porcine intrinsic cardiac neurons are concentrated in 11 distinct atrial and ventricular ganglionated plexuses. These extensive plexuses, along with fewer scattered neurons, display varied neuronal morphology and synaptology that represent the anatomical substrate for complex information processing within the intrinsic cardiac component of the porcine cardiac neuronal hierarchy. These anatomical data provide a framework for physiological analyses of the porcine intrinsic cardiac nervous system.

A p53 homologue and a novel serine proteinase inhibitor are over-expressed in lung squamous cell carcinoma.

LSCC is a common type of lung cancer and accounts for approximately 30% of all lung cancers. We have used a combination of subtraction and cDNA microarray technology to identify genes preferentially over-expressed in LSCC. Here we report extensive molecular characterization of two novel full-length cDNA sequences, L530S and L531S. Although L530S and L531S were found to be differentially over-expressed in LSCC, the expression profiles for these two genes were not identical. L530S expression was specifically elevated in LSCC whereas L531S transcript was up regulated in both LSCC and head and neck squamous cell carcinoma samples. L530S is a homologue of p53, and L531S belongs to a new member of serine proteinase inhibitors with significant homology to SCCA1 and SCCA2. Furthermore, L531S protein was found to be expressed in lung cancers by IHC analysis. The distinct as well as similar expression profiles exhibited by L530S and L531S suggest that each gene may play a unique role for tumorgenesis of LSCC. Identification of these genes not only allows us to further explore their diagnostic and therapeutic potentials for LSCC, but also provides us with additional tools and reagents for understanding the biology behind LSCC, and differentiating LSCC from other types of lung cancer at the molecular level.

Transient resistance of influenza virus to interferon action attributed to random multiple packaging and activity of NS genes.

Interferon (IFN) action survival curves for an avian influenza virus (AIV) in chicken or quail cells showed that 40-60% of the virions in a stock of virus were highly sensitive to the inhibitory effects of chicken IFN-alpha (ChIFN-alpha), whereas the rest were up to 100 times less sensitive. This greater resistance to IFN was transient, that is, was not a stable characteristic, in that virus stocks grown from plaques that formed in the presence of 50-800 U/ml IFN gave rise to virus populations that contained both sensitive and resistant virions. If AIV was serially passaged several times in the presence of IFN, the proportion of transiently IFN-resistant virus was greater. We propose a model to account for this transient resistance of AIV to IFN action based on the reported inactivation of the dsRNA-dependent protein kinase (PKR) and its activator dsRNA by the NS1 protein of influenza virus and also on the increase in the survival of AIV in IFN-treated cells exposed to 2-aminopurine, a known inhibitor of PKR. We suggest that IFN-resistant AIV is generated from a random packaging event that results in virions that contain two or more copies of RNA segment 8, the gene segment that encodes the NS1 protein of AIV, and that these virions will produce correspondingly elevated levels of NS1. The experimental data fit well to theoretical curves based on this model and constructed from the fraction of virus in the population expected by chance to contain one, two, or three copies of the NS gene when packaging an average of 12 influenza gene segments that include the 8 segments essential for infectivity.

Hyperthermic induction of the 27-kDa heat shock protein (Hsp27) in neuroglia and neurons of the rat central nervous system.

The 27-kDa heat shock protein (Hsp27) is constitutively expressed in many neurons of the brainstem and spinal cord, is strongly induced in glial cells in response to ischemia, seizures, or spreading depression, and is selectively induced in neurons after axotomy. Here, the expression of Hsp27 was examined in brains of adult rats from 1.5 hours to 6 days after brief hyperthermic stress (core body temperature of 42 degrees C for 15 minutes). Twenty-four hours following hyperthermia, Western blot analysis showed that Hsp27 was elevated in the cerebral cortex, hippocampus, cerebellum, and brainstem. Immunohistochemistry for Hsp27 revealed a time-dependent, but transient, increase in the level of Hsp27 immunoreactivity (Hsp27 IR) in neuroglia and neurons. Hsp27 IR was detected in astrocytes throughout the brain and in Bergmann glia of the cerebellum from 3 hours to 6 days following heat shock. Peak levels were apparent at 24 hours, gradually declining thereafter. In addition, increases in Hsp27 IR were detected in the ependyma and choroid plexus. Hyperthermia induced Hsp27 IR in neurons of the subfornical organ and the area postrema within 3 hours and reached a maximum by 24 hours with a return to control levels 4-6 days after hyperthermia. Specific populations of hypothalamic neurons also showed Hsp27 IR after hyperthermia. These results demonstrate that hyperthermia induces transient expression of Hsp27 in several types of neuroglia and specific populations of neurons. The pattern of induced Hsp27 IR suggests that some of the activated cells are involved in physiological responses related to body fluid homeostasis and temperature regulation.

Pathology of intrinsic cardiac neurons from ischemic human hearts.

Various populations of intrinsic cardiac neurons influence regional cardiac function tonically. It is not known whether such neurons are affected by disease states and, if so, in what manner. Therefore, the morphology of intrinsic cardiac ganglia obtained from patients with angiographic evidence of compromised regional coronary blood supply was studied. Posterior atrial ganglia and surrounding fat, removed at the time of cardiac surgery, were placed immediately in saline and within 15-120 min (average of about 40 min) in 0.5% paraformaldehyde/2.5% glutaraldehyde. In 32 studied ganglia, 35% of 473 intrinsic cardiac neurons displayed striking pathological changes at the light and ultrastructural level. The other cells displayed normal morphology. The cytoplasm of 74% of the abnormal cells had one or more of three types of inclusions: (1) darkly stained lamellated inclusions (Type I), (2) membrane-bound whorls and parallel arrays of lightly stained membranes, as well as fine granular material (Type II), or (3) concentric layers of lightly stained membranes with a darker, granular core (Type III). Neurons with inclusions were markedly enlarged (66 x 54 microm vs. 40 x 34 microm for normal neurons) and displayed fewer dendrites. Some neurons contained electron lucent vacuoles indicative of degeneration while others showed frank degeneration, being fragmented, shrunken, and misshapen. Phagocytic cells containing lamellated inclusions and cellular debris were found in ganglia with abnormal neurons. Some axon terminals also displayed degenerative changes. The identification of pathological changes in the human intrinsic cardiac nervous system has implications with respect to the functional integrity of this final common regulator of cardiac function in disease states.

Efferent and collateral organization of paratrigeminal nucleus projections: an anterograde and retrograde fluorescent tracer study in the rat.

The paratrigeminal nucleus (PTN) receives primary visceral afferent projections through cranial nerves IX and X and somatic afferent projections through cranial nerve V and dorsal roots as far caudally as C7. Pressure injections of the anterograde tracer tetramethylrhodamine dextran into the PTN in the rat resulted in bilateral labeling in the nucleus of the tractus solitarius, dorsal motor nucleus of the vagus nerve, and parabrachial nucleus. Anterograde labeling in the parabrachial nucleus was strongest in the external medial, external lateral, and ventral lateral subnuclei. Anterograde labeling was also found in the contralateral paratrigeminal nucleus, lamina I of the spinal trigeminal nucleus subnucleus caudalis, and ventroposteromedial nucleus of the thalamus. The collateral organization of PTN neurons was demonstrated by injecting different fluorescent retrograde tracers into the terminal fields of PTN projections as determined by the anterograde tracing experiments. Double-labeled neurons were found in the paratrigeminal nucleus following all combinations of injection sites. The most prominent PTN efferent projections and the most highly collateralized were to the nucleus of the tractus solitarius and parabrachial nucleus. The efferent and collateral connections of the paratrigeminal nucleus may provide a neuroanatomical substrate for integrating convergent visceral and somatic afferent information used to modulate autonomic function and behavior related to thermoregulation, nociception, and gustation.

Induction of the 27-kDa heat shock protein (Hsp27) in the rat medulla oblongata after vagus nerve injury.

The 27-kDa heat shock protein (Hsp27) is constitutively expressed in motor and sensory neurons of the brainstem. Hsp27 is also rapidly induced in the nervous system following oxidative and cellular metabolic stress. In this study, we examined the distribution of Hsp27 in the rat medulla oblongata by means of immunohistochemistry after the vagus nerve was cut or crushed. After vagal injury, rats were allowed to survive for 6, 12, 24 h, 2, 4, 7, 10, 14, 30, or 90 days. Vagus nerve lesions resulted in a time-dependent up-regulation of Hsp27 in vagal motor and nodose ganglion sensory neurons that expressed Hsp27 constitutively and de novo induction in neurons that did not express Hsp27 constitutively. In the dorsal motor nucleus of the vagus nerve (DMV) and nucleus ambiguus, the levels of Hsp27 in motor neurons were elevated within 24 h of injury and persisted for up to 90 days. Vagal afferents to the nucleus of the tractus solitarius (NTS) and area postrema showed increases in Hsp27 levels within 4 days that were still present 90 days postinjury. In addition, increases in Hsp27 staining of axons in the NTS and DMV suggest that vagus nerve injury resulted in sprouting of afferent axons and spread into areas of the dorsal vagal complex not normally innervated by the vagus. Our observations are consistent with the possibility that Hsp27 plays a role in long-term survival of distinct subpopulations of injured vagal motor and sensory neurons.

Cholinesterases in cardiac ganglia and modulation of canine intrinsic cardiac neuronal activity.

Cholinergic neurotransmission plays a significant role in intrinsic cardiac ganglia with the action of acetylcholine being terminated by acetylcholinesterase (AChE, EC 3.1.1.7). Anatomical studies were performed to characterize neurons associated with AChE and a closely related enzyme, butyrylcholinesterase (BuChE, EC 3.1.1.8), in canine intrinsic cardiac ganglia. Histochemical staining for AChE and BuChE in canine right atrial neurons showed that there were four neuronal populations, namely, those that contained AChE only, BuChE only, both AChE and BuChE, and those that did not contain either enzymes. The neuronal activity of intrinsic cardiac neurons in response to substrates and inhibitors of cholinesterases were studied in anesthetized dogs. The activity of intrinsic cardiac neurons, as measured by changes in the number of action potentials, increased by local application of acetylcholine. However, local application of butyrylcholine led to a considerably greater increase in the activity of intrinsic cardiac neurons. In keeping with the neurochemical heterogeneity in intrinsic cardiac ganglia with respect to cholinesterases, the activity generated by most butyrylcholine-sensitive neurons was not influenced by acetylcholine and the activity generated by the most acetylcholine-sensitive neurons was not influenced by butyrylcholine. This suggests that these two agents preferentially influence different populations of intrinsic cardiac neurons. Enzyme kinetic studies demonstrated that canine AChE preferentially catalyzed the hydrolysis of acetylcholine while canine BuChE preferentially catalyzed the hydrolysis of butyrylcholine. Cholinesterase inhibitors Ro 2-1250 and Ro 2-0638 inhibited both canine cholinesterases, while huperzine A preferentially inhibited canine AChE and ethopropazine inhibited canine BuChE. The activity of neurons in the intrinsic cardiac ganglia significantly increased when Ro 2-1250 or Ro 2-0638 was administered locally. The activity of neurons was not affected when huperzine A or ethopropazine was administered, indicating that both cholinesterases must be inhibited to increase neuronal activity. In summary, these data show that in addition to AChE, intrinsic cardiac ganglia also contain distinct populations of neurons that are associated with BuChE, and the activity generated by these neurons is differentially influenced by their substrates. Because simultaneous inhibition of AChE and BuChE leads to increased neuronal activity, it is concluded that AChE- and BuChE-positive intrinsic cardiac neurons may act synergistically to influence the overall tonic activity of intrinsic cardiac ganglia.

Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain.

The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide-cyclic GMP signal transduction pathway in virtually every area of the rat brain.

Convergent prefrontal cortex and mamillary body projections to the medial pontine nuclei: a light and electron microscopic study in the rat.

To study the convergence of medial prefrontal cortex and mamillary body projections to the medial pontine nuclei, light and electron microscopic, neuroanatomical, tract-tracing experiments were performed. Injections of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP), biotin conjugated to dextran (BD), or rhodamine conjugated to dextran (RD) were made individually or in combinations into the cerebral cortex, hypothalamus, or pons. In addition, injections of WGA-HRP into the medial prefrontal cortex and electrolytic lesions of the mamillary body were made to study the synaptology of afferent projections to the pontine nuclei. In the light microscopic studies, injections of WGA-HRP into the rostromedial pontine nuclei produced dense, retrograde labeling both in the dorsal peduncular area of the medial prefrontal cortex and in the medial mamillary nucleus, pars medialis. Injections of the anterograde tracers BD and RD into the medial prefrontal cortex and the medial mamillary nuclei, respectively, resulted in partially overlapping terminal fields in the rostromedial pontine nuclei. In the electron microscopic studies, injections of WGA-HRP into the dorsal peduncular area and electrolytic lesions of the mamillary body produced anterogradely labeled axon terminals and degenerating axon terminals that synapsed on the same dendrites or neuronal somata in the rostromedial pontine nuclei. The results demonstrate that the medial prefrontal cortex and the medial mamillary nuclei have partially overlapping projections to the rostromedial pontine nuclei and implicate precerebellar relay nuclei in the integration of limbic and/or autonomic functions mediated by convergent projections from the cerebral cortex and the hypothalamus.

Neurochemical organization of paratrigeminal nucleus projections to the dorsal vagal complex in the rat.

The paratrigeminal nucleus, located in the spinal trigeminal tract rostral to the obex, is important in the integration of visceral and somatosensory afferent information and may modulate autonomic function through its projections to the dorsal vagal complex. Anterograde and retrograde neuroanatomical tracers were used in conjunction with immunohistochemistry to determine the neurochemical organization of the efferent pathway from the paratrigeminal nucleus to the dorsal vagal complex in the rat. Double-labelling studies demonstrated that leu-enkephalin, 28-kDa calbindin, and neuronal nitric oxide synthase were present in neurons in the paratrigeminal nucleus that project to the dorsal vagal complex. The results of this study are consistent with the hypothesis that neurochemically distinct pathways from the paratrigeminal nucleus are involved in the sensory modulation of autonomic function.

Nitric oxide-mediated cGMP production in the islands of Calleja in the rat.

cGMP-immunostaining in the islands of Calleja (ICj) in slices incubated in vitro partially co-localized with nitric oxide synthase (NOS) inside the ICj. No cGMP-immunostaining was found outside the ICj in unstimulated slices, whereas the NO-donor sodium nitroprusside (SNP) stimulated cGMP in cells and fibers bordering on the ICj. These findings show an ongoing NO synthesis in in vitro slices and suggest a relatively restricted diffusion range for endogenously synthesized NO.

Distribution of butyrylcholinesterase in the human amygdala and hippocampal formation.

The distribution of the major cholinergic regulatory enzyme acetylcholinesterase (AChE, EC 3.1.1.7) has been extensively studied in the human brain, but the distribution of the closely related enzyme butyrylcholinesterase (BuChE, EC 3.1.1.8) is largely unknown. Because of the importance of BuChE and AChE in Alzheimer's disease, we have studied the distribution of BuChE in the normal human amygdala and hippocampal formation and compared it with that of AChE by using histochemical techniques. In the amygdala, the distribution of BuChE differed significantly from that of AChE in that BuChE was found primarily in neurons and their dendritic processes, whereas AChE was found predominantly in the neuropil. BuChE-positive neurons were present in up to 10% of the neuronal profiles in lateral, basolateral (basal), basomedial (accessory basal), central, cortical, and medial amygdaloid nuclei. AChE was found primarily in the neuropil in these nuclei with only a few AChE-positive neurons. In the hippocampal formation, BuChE was also found in neurons and not in the neuropil, whereas AChE was found in both neurons and in the neuropil. BuChE and AChE neurons were present in the polymorphic layer of the dentate gyrus, as well as the stratum oriens and stratum pyramidale of the hippocampus proper. There was considerable overlap in shapes, sizes, and numbers of BuChE- and AChE-positive neurons, suggesting that the enzymes were colocalized in neurons of the hippocampal formation. The distinct distribution of BuChE suggests that it may have specific functions including coregulation of cholinergic and noncholinergic neurotransmission in human amygdala and hippocampal formation.

Brainstem cells of origin of physiologically identified cardiopulmonary nerves in the rhesus monkey (Macaca mulatta).

The distributions of brainstem cells of origin of the cervical vagus nerve, its cervical and thoracic branches, and of neurons projecting to the cricothyroid muscle and the stomach wall were identified and compared following injections of horseradish peroxidase (HRP) in 18 Rhesus monkeys. Physiologically and/or anatomically identified cardiopulmonary nerves were injected with 3-20 microl of HRP to identify the locations of vagal preganglionic cardioinhibitory neurons in 10 of these monkeys. After injections into cardiopulmonary nerves, retrogradely labelled cells were concentrated ipsilaterally in the most lateral parts of the dorsal motor nucleus of the vagus nerve (DMV) and in the ventrolateral nucleus ambiguus (NA). Fewer labelled neurons were identified close to or in the principal (dorsal) division of the NA and in the intermediate zone between the DMV and NA. The results indicate that monkey cardiopulmonary nerves have multiple origins; their somata are located primarily in the ventrolateral NA and to a lesser extent in the lateral DMV. In monkeys, there is a stronger representation in the lateral DMV than in cat, dog and pig. The viscerotopic organization of the cells of origin of primate vagal nerves is similar to that in other species. The cells of origin of vagal projections to the superior laryngeal nerve and cricothyroid muscle were located in the NA rostrally to those of the inferior laryngeal nerve. Injections into the superior laryngeal nerve also resulted in significant labelling in the DMV and intermediate zone (IZ). The cells of origin of projections to the anterior stomach wall were restricted to the DMV with a bilateral distribution of labelled cells, concentrated medially in the nucleus.

Regulation of interferon-induced protein kinase PKR: modulation of P58IPK inhibitory function by a novel protein, P52rIPK.

The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways. Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function. The interferon-induced serine/threonine protein kinase, PKR, is activated in response to various environmental stimuli. Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58IPK, a cellular PKR inhibitor. P58IPK functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2alpha subunit (eIF-2alpha) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth. To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58IPK-interacting protein. This protein, designated P52rIPK, possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines. P52rIPK and P58IPK interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts. When coexpressed with PKR in yeast, P58IPK repressed PKR-mediated eIF-2alpha phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function. Conversely, introduction of P52rIPK into these strains resulted in restoration of both PKR activity and eIF-2alpha phosphorylation, concomitant with growth suppression due to inhibition of P58IPK function. Furthermore, P52rIPK inhibited P58IPK function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58IPK is inhibited through a direct interaction with P52rIPK which, in turn, results in upregulation of PKR activity. Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways.