Partial cloning of putative G-proteins modulating mechanotransduction in the ciliate stentor.

Signal transduction systems known to utilize G-proteins in higher eukaryotes undoubtedly evolved prior to the development of metazoa. Pharmacological evidence indicates that the ciliates Paramecium, Stentor, and Tetrahymena all utilize signaling systems similar to those found in mammals. However, there has been relatively little direct evidence for the existence of G-proteins in ciliates. Since highly conserved heterotrimeric G-proteins form the basis of receptor-coupled signal transduction systems in a wide variety of metazoa, it is of interest to know if these important signaling molecules were early to evolve and are present and functionally important in a wide variety of unicellular organisms. We have previously shown that mechanotransduction in Stentor is modulated by opiates in a manner that may involve pertussis toxin-sensitive G-proteins. Here we utilize drugs known to interact with G-proteins to further test for the involvement of these important signaling molecules in Stentor mechanotransduction. We present behavioral and electrophysiological data demonstrating that putative G-proteins in Stentor decrease mechanical sensitivity by modulating the mechanotransduction process. In addition, we report the partial cloning of 4 G-protein alpha-subunits from Stentor. We confirm that these clones are of Stentor origin and are transcribed. Furthermore, we employ antisense oligodeoxynucleotide-mediated knockout to demonstrate that these ciliate G-proteins exert a modulatory influence on Stentor behavior, and that a G1/G0-like clone mediates the inhibitory action of beta-endorphin on mechanotransduction.

Suckling stimulus suppresses messenger RNA for tyrosine hydroxylase in arcuate neurons during lactation.

Tyrosine hydroxylase (TH) mRNA in tuberoinfundibular dopamine (TIDA) neurons is suppressed during lactation but rebounds upon pup removal. A time course of TH mRNA changes after pup removal revealed three phases: (1) a nuclear phase (evident 1.5 hours after pup removal, maximal at 3 hours) with TH mRNA appearing in 1 or 2 nuclear loci with little or no change in cytoplasmic mRNA; (2) a cytoplasmic phase (noted 6 hours after pup removal, peaked 12-24 hours) with a significant increase in total TH mRNA levels mainly in the cytoplasm; and (3) a stabilization phase (24-48 hours after pup removal) when nuclear signals were low and cytoplasmic RNA showed a slight decline with extension of RNA clusters into the cell dendrites. In rats whose pups could suckle only on one side, TH was up-regulated only on the side contralateral to nipple blockade. These data indicate that after suckling terminates, TH up-regulation is evident at 1.5 hours, but 6 hours is needed before the cells transport sufficient mRNA into the cytoplasm. The rapid signaling of TH up-regulation stems from the fact that the TIDA neurons respond to neural signals from termination of suckling.

Analysis of tyrosine hydroxylase gene transcription using an intron specific probe.

The nuclear run-on assay is the most commonly used technique to determine transcription rates of specific genes such as tyrosine hydroxylase. Its application to studies in the nervous system is problematic, however, as a result of limitations in sensitivity and the loss of anatomical integrity. We observed that the relative levels of tyrosine hydroxylase intron 2-containing RNA using a ribonuclease protection assay in the adrenal medulla changed in response to pharmacological treatments consistently with changes shown by the nuclear run-on assay. Our results indicate that measures of tyrosine hydroxylase primary transcript levels offer an alternative to the nuclear run-on assay and validate the application of intron-specific in situ hybridization as a means of assessing the relative transcriptional activity of the tyrosine hydroxylase gene. Similar quantitative results were obtained using intron-specific in situ hybridization with oligonucleotide probes specific for rat tyrosine hydroxylase intron 2. Furthermore, we observed that intron-specific in situ hybridization could be used to measure tyrosine hydroxylase transcription rates in the locus coeruleus, providing resolution at the level of single neurons. Thus, measuring the levels of tyrosine hydroxylase intron 2 provides a sensitive measure of tyrosine hydroxylase transcription rate that can be applied to the study of brain catecholaminergic neurons.

Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA.

Tissue-and stage-specific pre-mRNA splicing events are prevalent in mammals, yet molecular details are lacking about these important mechanisms of posttranscriptional gene control. In this study, we investigate the regulated splicing of rat gamma 2 pre-mRNA, a subunit of the GABAA receptor, as a step toward understanding the molecular basis of a neuron-specific splicing event involving cassette exon selection. Cell-and substrate-specific regulation of gamma 2 pre-mRNA is recapitulated in a neuronal cell line derived from the cerebellum, which produces enhanced levels of the exon-selected mRNA. In contrast, a control cell line derived from non-neuronal cells of the pituitary produces prominent levels of the unregulated, exon-skipped mRNA. The cerebellar and pituitary cell lines are well matched in overall splicing efficiency and produce an invariant pattern of splicing for a control substrate, which is alternatively spliced but not regulated in this system. The appropriateness of the two cell lines is indicated by an extended mRNA mapping experiment, which documents the region-specific switch in exon selection throughout rat brain. Using this pair of cell lines, we show that large intron segments flanking the regulated exon are dispensable for regulation. These intron regions have been deleted to generate a minimal splicing substrate for the purpose of identifying essential RNA elements. In this context, we show that essential nucleotides are located at positions +7, +8, and +9 of the regulated exon and in a 9-nt adenosine-rich region of the adjacent 3' splice site. Due to the proximity and base complementarity of the required nucleotides, experiments were devised to test models involving the recognition of two single-stranded signals, or one duplex RNA signal. These results clearly disfavor the duplex RNA recognition model and indicate that the required regions are recognized as independent, single strands in neuronal cells. A weak 5' splice site adjacent to the regulated exon is required as a third essential element. Although the importance of a weak 5' splice site is common to other regulated systems such as NCAM, the essential nucleotides in the exon and 3' splice site region defined in this study for gamma 2 splicing regulation are novel.

Dexamethasone and stress upregulate Kv1.5 K+ channel gene expression in rat ventricular myocytes.

Hormones may produce long-term effects on excitability by regulating K+ channel gene expression. Previous studies demonstrated that administration of dexamethasone, a glucocorticoid receptor agonist, to adrenalectomized rats, rapidly induces Kv1.5 K+ channel expression in the ventricle of the hear. Here, RNase protection assays and Northern blots are used to examine the cell type specificity of dexamethasone action and to test whether Kv1.5 gene expression can be regulated by a physiological stimulus. We show that Kv1.5 mRNA expression in the central nervous system is highest in the hypothalamus. However, dexamethasone treatment of adrenalectomized rats fails to affect Kv1.5 mRNA levels in hypothalamus or lung. In contrast, dramatic upregulation of Kv1.5 mRNA is seen in skeletal muscle and pituitary. Increased Kv1.5 message also found in isolated ventricular cardiomyocytes following in vivo treatment with dexamethasone. Finally, it is shown that cold stress of intact rats significantly increases cardiac Kv1.5 mRNA expression. We conclude that dexamethasone induction of Kv1.5 gene is tissue-specific. Furthermore, our results suggest that stress may act via glucocorticoids to increase Kv1.5 gene expression in ventricular cardiomyocytes. Hence, K+ channel gene expression can be influenced by physiological and pharmacological stimuli.

Alterations in tyrosine hydroxylase expression following partial lesions of the nigrostriatal bundle.

Effects of destruction of central dopaminergic neurons on tyrosine hydroxylase gene expression were investigated. Two weeks after the unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, a 67% to 99% loss of striatal dopamine (DA) content was observed ipsilateral to the injection site. Measures of tyrosine hydroxylase (TH) protein levels revealed losses in striatal content proportional to DA content. Striatal dihydroxylphenylacetic acid (DOPAC) was somewhat less affected, resulting in 2- to 4-fold increases in the striatal DOPAC/DA ratio, depending on the severity of the lesion. Morphologically, surviving TH-positive substantia nigra pars compacta (SNc) neurons were more rounded than contralateral control cells, and exhibited decreases in cross-sectional area that were proportional to the loss of striatal DA. Measures of cytoplasmic TH mRNA levels in surviving neurons by in situ hybridization autoradiography revealed a significant 23% decrease in TH content per cell that could be correlated to lesion size. The decreases in cross-sectional area and TH mRNA content resulted in a small decrease in TH mRNA density of 6%. The determination of TH transcription rate by an intron-directed in situ hybridization assay found no significant change in TH transcriptional activity as a function of lesion. We conclude that the short-term effect of partial 6-OHDA-induced lesions of the nigrostriatal dopaminergic pathway is the selective loss or shrinkage of large DA neurons of the SNc, and that the associated down-regulation of TH mRNA expression in surviving neurons is due to a post-transcriptional mechanism related either to concomitant cellular hyperactivity or is secondary to the morphological alterations.

Induction of glucocorticoid receptor expression in hypothalamic magnocellular vasopressin neurons during chronic hypoosmolality.

The site of action for the modulation of hypothalamo-neurohypophyseal function by adrenal steroids is not known. Glucocorticoid receptors (GR) are an obvious potential site of action, but there have been conflicting reports of GR localization in magnocellular neurons. We confirm studies finding undetectable levels of GR in vasopressin or oxytocin neurons, but now report that chronic hypoosmolality induces the expression of GR in magnocellular vasopressin neurons, but not oxytocin neurons. These data support the hypothesis that the vasopressin gene can be directly inhibited by glucocorticoids, and that the induction of GR expression in magnocellular neurons may be part of a redundant set of mechanisms to suppress the expression of AVP during periods of prolonged hypoosmolality. This mechanism represents a novel form of steroid feedback control in brain.

Models of neurohypophyseal homeostasis.

Rats subject to prolonged (3-6 days) hypernatremia show significantly decreased pituitary vasopressin content as well as increased levels of hypothalamic vasopressin mRNA; these values return to baseline levels after the stimulus is removed. In this paper, we tested whether a single cellular mechanism for regulation of synthesis could account for the experimental observations of both pituitary hormone depletion-repletion and hypothalamic mRNA content. We developed several "minimal" models of vasopressin synthesis in which control of hormone synthesis was regulated exclusively by transcription, translation, or mRNA decay and tested each model to see which best emulated the dynamics of neuro-hypophyseal vasopressin content and hypothalamic vasopressin mRNA. Experimental data provided parameters for pituitary content, baseline and stimulated release rates, mRNA decay, transcription, and translation. Models based exclusively on translation and mRNA decay failed to produce predictions similar to experimental observations. Of the models tested, the transcription model provided predictions most consistent with laboratory data, although some quantitative differences remain. The results of the computer modeling strongly suggest that transcription represents the predominant means by which magnocellular neurons regulate vasopressin synthesis.

In situ hybridization analysis of arginine vasopressin gene transcription using intron-specific probes.

In situ hybridization histochemistry with a probe directed against an intron sequence of the rat arginine vasopressin (AVP) gene was used to demonstrate localization and regulation of AVP heteronuclear RNA in discrete brain regions. Hybridization with an AVP intron I (AVPinI) probe revealed specific hybridization confined to cell nuclei of paraventricular nucleus, supraoptic nucleus (SON), and suprachiasmatic nucleus neurons of the rat hypothalamus. Grain counts revealed that the signal generated by the AVPinI probe represented 1.9% of that derived from an AVP exon C probe (AVPexC) in the SON. Interestingly, in the suprachiasmatic nucleus the proportion of AVPinI to AVP exon C ratio was much higher (12%), suggesting either increased transcription of the AVP gene or changes in posttranscriptional RNA processing. Regulatory experiments revealed that 2.6-fold increases in AVPinI signal could be visualized in the SON as little as 30 min after an acute salt load, a period during which no significant change in cytoplasmic AVP mRNA could be observed. In response to chronic salt loading, both AVP heteronuclear RNA and AVP mRNA were up-regulated. These data compared favorably with transcription rate values determined by nuclear run-on assay, suggesting that intronic in situ hybridization affords a relatively reliable method for assessment of rapid changes in gene transcription in individual central nervous system neurons.

Hyponatremia in rats induces downregulation of vasopressin synthesis.

Hyponatremia due to inappropriate secretion of vasopressin is a common disorder in human pathophysiology, but vasopressin synthesis during hypoosmolality has not been investigated. We used a new method to quantitate synthesis of vasopressin in rats after 3, 7, and 14 d of hyponatremia induced by administering dDAVP (a vasopressin agonist) and a liquid diet. Vasopressin synthesis was completely turned off by 7 d. Vasopressin mRNA levels in the hypothalamus paralleled the reduction in synthesis and were reduced to levels of only 10-15% of the content in control rats. When hyponatremia was corrected by withdrawal of dDAVP, vasopressin mRNA slowly returned to normal over 7 d. The observation that vasopressin synthesis can be so completely turned off leads to several conclusions: under normal physiological conditions the neurohypophysis is chronically upregulated; there must be an osmotic threshold for initiation of vasopressin synthesis (and release); the large store of hormone in the posterior pituitary is essential for vasopressin to be available during times of decreased synthesis; and, finally, some nonosmolar stimulus for synthesis must be present during clinical disorders when vasopressin is secreted (and synthesized) despite hypoosmolality.

Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus.

Rat brain expresses two types of corticosteroid-binding proteins. The type I receptor binds corticosterone with high affinity and is structurally related to the kidney mineralocorticoid receptor (MR), while the type II or classical glucocorticoid receptor binds corticosterone with lower affinity and displays an in vivo preference for dexamethasone. Here we describe the isolation and characterization of a cDNA coding for the MR, from a rat hippocampus cDNA library, by low stringency hybridization to radiolabeled human glucocorticoid receptor cDNA. The nucleotide and deduced amino acid sequence for rat hippocampal MR displays extensive homology to a MR cDNA isolated from human kidney, suggesting that they are orthologous genes. Southern analysis suggests that there is only one gene for the MR, and in vitro expression of the receptor generates a high affinity corticosterone-binding protein. These data provide evidence to support the contention that a single gene gives rise to the MR in renal tissues and type I receptors in the brain.

Characterization of pro-opiomelanocortin cDNA from the Old World monkey, Macaca nemestrina.

An observation from high-pressure liquid chromatography (HPLC) suggesting that monkey beta-endorphin (BE) was chemically different from human or rat BE was investigated by determining the cDNA sequence for the monkey pro-opiomelanocortin (POMC) precursor. A full-length cDNA for POMC was isolated from a Macaca nemestrina whole pituitary cDNA library. The longest open reading frame predicts a 264-residue polypeptide exhibiting the basic structure of POMC that is closely homologous to the human counterpart. The monkey BE sequence apparently diverged from the human sequence after the latter had made the His-27 to Tyr-27 change but prior to the Gln-31 to Glu-31 transition, leaving it more hydrophobic than rat or human BE, consistent with its chromatography on reverse-phase HPLC. Comparison of the monkey POMC precursor with those of other species highlights conserved domains, presumably reflecting regions of physiological activity that await elucidation.

Differential expression of vasopressin alleles in the Brattleboro heterozygote.

A solution hybridization/RNase protection assay for the molar quantitation of vasopressin and oxytocin mRNAs, using synthetic complementary RNA probes, is described. This assay was optimized to permit the identification of vasopressin (AVP) mRNAs containing the frame-shift point deletion causing inheritable diabetes insipidus in the Brattleboro strain of rat. Examination of RNA from hypothalamic magnocellular tissue punches found that of the 86.1 x 10(-18) mol [86.1 attomoles (amol)] of AVP mRNA detected in the Brattleboro heterozygote paraventricular (PVN) nucleus, 5.2% could be shown to be mutant AVP mRNA (AVPd RNA). The percentage of AVPd RNA increased dramatically to 18.1% after 6 d of chronic intermittent salt-loading. Similar percentages and percentage increases of AVPd RNA were detected in the heterozygote supraoptic nucleus (SON). These values were contrasted with those found in parallel studies in both Long Evans and Brattleboro homozygotes, and compared with values for oxytocin (OT) mRNA in all 3 AVP rat genotypes. The results of continued osmotic regulation of the mutant AVP gene, the low native levels of AVPd RNA found in both the Brattleboro heterozygote and homozygote, and the magnitudes of AVPd expression change with chronic osmotic challenge were interpreted as indicating that (1) in the diploid rat genome, both AVP alleles are transcribed, (2) the osmotic regulation of the mutant AVP gene is normal, and (3) the low levels of AVPd mRNA are consistent with a shorter-than-control effective mRNA half-life.

Regulation of hypothalamic magnocellular neuropeptides and their mRNAs in the Brattleboro rat: coordinate responses to further osmotic challenge.

A paradigm was developed for the chronic osmotic stimulation of homozygous diabetes insipidus rats of the Brattleboro strain, a strain that fails to synthesize vasopressin. This study examines the adaptation of 2 sets of coexisting peptide hormone magnocellular neurons in the hypothalamoneurohypophyseal system (HNS) of Long Evans (LE), Brattleboro heterozygote (HZ), and Brattleboro homozygote (DI) rats: (1) the arginine8-vasopressin (AVP)/dynorphin (DYN) neurons, and (2) the oxytocin (OT)/cholecystokinin (CCK8) neurons of the paraventricular and supraoptic nuclei, which project to the posterior pituitary. The regimen of chronic intermittent salt-loading (CISL) involved the replacement of 2% saline for normal drinking water for 18 hr/d. This protocol effectively increased plasma levels of AVP and OT in LE and HZ rats, oxytocin in DI rats, and maintained the posterior pituitary in a state depleted of AVP, OT, CCK, and peptides derived from pro-dynorphin: DYN A 1-17, DYN A 1-8, and DYN B 1-13. The ratio of pituitary DYN A 1-17 to DYN A 1-8 content in DI rats or in LE, HZ, and DI rats following 6 d of CISL suggests a preferential release of DYN A 1-17 during periods of chronic secretory activity. In response to chronic secretory activity, mRNAs for AVP, OT, DYN, and CCK increased 1.5-2-fold in all 3 AVP rat strains, with mRNAs for coexisting peptide hormones displaying parallel increases. Mutant AVP mRNA in the DI rat was expressed at very low levels and DYN mRNA in very high levels, with each of these mRNAs continuing to be regulated by CISL in a normal manner. These results suggest a regulatory relationship between AVP and OT neurons, in which vasopressin neurons are feedback-regulated by AVP, most likely via plasma osmolarity, and that oxytocin neurons are modulated by peptides derived from pro-dynorphin.

Pro-dynorphin is endogenous to the anterior pituitary and is co-localized with LH and FSH in the gonadotrophs.

Pro-dynorphin peptides have been shown to exist in the anterior lobe of the pituitary gland. The dynorphin in the anterior lobe is distinct from that which is co-localized with vasopressin in the magnocellular system in both post-translational processing and regulation of release. Here, we report on the existence of pro-dynorphin mRNA, approximately 2400 nucleotides in length, in the anterior lobe. Furthermore, we present immunocytochemical evidence for the co-existence of dynorphin, LH and FSH in a subset of gonadotrophs. These findings suggest a possible role of pro-dynorphin products in the regulation of the hypothalamic-pituitary-gonadal axis.

Hypothalamic dynorphin and vasopressin mRNA expression in normal and Brattleboro rats.

Peptides derived from prodynorphin and provasopressin precursors coexist within neurosecretory vesicles of magnocellular neurons of the rat hypothalamus projecting to the posterior pituitary. The secretory activity of these neurons can be stimulated with physiological manipulations that elevate plasma levels of vasopressin (VP), such as dehydration and salt-loading. Evidence indicates that both VP- and prodynorphin-derived peptides are secreted under such conditions. With chronic osmotic challenge, the mRNAs for both prodynorphin and provasopressin increase in parallel in the supraoptic and paraventricular nuclei of the hypothalamus, and not within nonmagnocellular cell groups projecting elsewhere in the brain. The results indicate an example of coordinate regulation of mRNA expression for coexisting peptides within the brain. These results from microdissected tissues have been coupled with the more anatomically precise method of in situ hybridization histochemistry. Using 35S-radiolabeled synthetic oligonucleotides complementary to VP and dynorphin mRNAs, these mRNAs have been autoradiographically localized to magnocellular parikarya in the rat hypothalamus. Results also indicate that this technology can be used for regulatory studies, as evidenced by the increased hybridization of VP oligonucleotide to hypothalamic nuclei from salt-loaded rats.

Rapid expression of novel proteins in goldfish retina following optic nerve crush.

Polyadenylated messenger RNA was isolated from goldfish retinas at various times following unilateral crush of the optic nerve. RNA was translated in a cell-free system and product proteins analyzed by two-dimensional gel electrophoresis and autofluorography. Poly(A)+ mRNA-directed protein synthesis revealed an 8-fold increase in the labeling of polypeptides of about 30 kd Mr and a pI of 5.5 in retinas 2 d following optic nerve crush, compared with control retina mRNA translation products. In vitro labeling of retinal proteins revealed the enhanced synthesis of comparable 30 kd proteins in 2 d post-crush retinas. Evidence presented suggests that this 30 kd protein cluster may correspond to fish 30 kd stress or heat-shock proteins (hsp-30).

Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe.

Synthetic oligonucleotide probes can be easily obtained and used, in contrast to cDNA cloning to develop probes, and thus the present study was carried out to determine whether such probes could also be useful for in situ hybridization. A 24-base synthetic oligonucleotide complementary to part of the alpha-melanocyte-stimulating hormone (alpha-MSH) coding region of proopiomelanocortin (POMC) mRNA was 5'-end-labeled by using [gamma-32P]ATP with T4 polynucleotide kinase or was 3' tailed by using [alpha-32P]dATP or [3H]dCTP with terminal deoxynucleotidyltransferase. Blot analysis of pituitary poly(A)+ RNA showed that the oligonucleotide hybridized to a single species with a molecular size of approximately 1200 nucleotides, consistent with that determined previously for POMC mRNA. The oligonucleotide, regardless of labeling method, hybridized to cells in the pituitary intermediate lobe, but not in the posterior lobe. Only the 3H-labeled probe gave resolution of individual pituitary anterior lobe cells. The specificity of the hybridization was determined by showing that the intermediate lobe signal was blocked by prehybridization of the tissue with unlabeled alpha-MSH oligonucleotide probe. Furthermore, the hybridized probe exhibited a sharp sigmoid curve when melted off. Finally, the oligonucleotide probe detected, in situ, the haloperidol-induced elevation of intermediate lobe POMC mRNA. Thus, the oligonucleotide probe exhibited hybridization in an anatomically and biochemically specific manner, and it detected a tissue-specific change in mRNA levels in situ.

Vasopressin mRNA regulation in individual hypothalamic nuclei: a northern and in situ hybridization analysis.

The present study examines the relative levels of vasopressin (AVP) mRNA within the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei of the rat hypothalamus, and details the rates at which these levels change over the course of a 6 d salt-loading regimen. The quantitation of vasopressin mRNA was achieved by using three different procedures: (1) cell-free translation in rabbit reticulocyte lysate or (2) Northern analysis of poly(A)RNAs isolated from micro-punch dissected SON, PVN, and SCN, and (3) in situ hybridization histochemistry. The former involved the quantitative immunoprecipitation of the neurophysin precursors containing arginine8-vasopressin (AVP) or oxytocin, and the latter two techniques employed a radiolabeled synthetic oligodeoxynucleotide complementary to the 3' region of the AVP mRNA. Both the cell-free studies and the Northern gel analyses detected a sevenfold increase of AVP mRNA in the SON, a fivefold increase in the PVN, and no significant change in the SCN following 6 d of salt-loading. After the initiation of salt-drinking, these increases were shown to occur between 24 and 48 hr in the SON and between 48 and 72 hr in the PVN. The in situ hybridization studies revealed the anatomically correct hybridization of either 32P- or 3H-labeled AVP oligonucleotide to magnocellular perikarya within both the SON and PVN. Autoradiographic grains could be shown to be confined to the cytoplasm of these cells, and could be co-localized with immunoreactivity directed against the carboxy terminus of the AVP percursor. Comparison of x-ray level autoradiograms of control and 6 day salt-loaded SON revealed up to a sevenfold increase in specific signal in the salt-loaded sections. It is concluded that the response of AVP mRNA to osmotic stimuli in the three hypothalamic nuclei is heterogeneous, and that this heterogeneity can be explained by separating AVP neurons into two systems: one responsible for eliciting the antidiuretic actions of AVP via plasma AVP levels, and the other involved in CNS activities not directly involved with antidiuresis.