DNA-excess Solution Hybridization Research Articles

Alpha-2-Adrenergic activation of proopiomelanocortin-containing neurons in the arcuate nucleus causes opioid-mediated hypotension and bradycardia.

Treatment of rats for 4 days with alpha-methyldopa, 200 mg/kg/day i.p., increases steady state levels of proopiomelanocortin (POMC) mRNA in the mediobasal hypothalamus, as measured by DNA excess solution hybridization. The increase is prevented by parallel treatment with yohimbine, 2 mg/kg/day i.p., but not by naltrexone, 2 mg/kg/day i.p. Treatment with the peripheral vasodilator hydralazine, 2 mg/kg/day, does not affect POMC mRNA levels. In situ hybridization histochemistry with a cRNA probe for POMC indicates that POMC-containing cells are located within the confines of the arcuate nucleus both in control and in alpha-methyldopa-treated rats, and confirms the increase in POMC mRNA in the latter. Microinjection of 2 micrograms of alpha-methylnorepinephrine unilaterally into the arcuate nucleus of urethane-anesthetized rats causes hypotension and bradycardia, which can be inhibited by 200 ng of yohimbine microinjected into the same site, or by 100 ng l-naloxone microinjected into the ipsilateral nucleus tractus solitarii, but not into the arcuate nucleus. These findings are interpreted to indicate that activation of alpha 2-adrenergic receptors located on POMC-containing neurons in the arcuate nucleus causes beta-endorphin release and stimulation of opiate receptors in the NTS, which results in hypotension and bradycardia, and that this mechanism contributes to the hypotensive action of alpha-methyldopa.

Glucocorticoids down-regulate beta 1-adrenergic-receptor expression by suppressing transcription of the receptor gene.

The expression of beta 2-adrenergic receptors is up-regulated by glucocorticoids. In contrast, beta 1-adrenergic receptors display glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of beta-adrenergic receptors, the synthetic glucocorticoid dexamethasone stimulates no change in the total beta-adrenergic receptor content, but rather shifts the beta 1:beta 2 ratio from 80:20 to 50:50. Radioligand binding and immunoblotting demonstrate a sharp decline in beta 1-adrenergic receptor expression. Metabolic labelling of cells with [35S]-methionine in tandem with immunoprecipitation by beta 1-adrenergic-receptor-specific antibodies reveals a sharp decline in the synthesis of the receptor within 48 h for cells challenged with glucocorticoid. Steady-state levels of beta 1-adrenergic-receptor mRNA declined from 0.47 to 0.26 amol/microgram of total cellular RNA within 2 h of dexamethasone challenge, as measured by DNA-excess solution hybridization. The stability of receptor mRNA was not influenced by glucocorticoid; the half-lives of the beta 1- and beta 2-subtype mRNAs were 1.7 and 1.5 h respectively. Nuclear run-on assays revealed the basis for the down-regulation of receptor expression, i.e. a sharp decline in the relative rate of transcription for the beta 1-adrenergic-receptor gene in nuclei from dexamethasone-treated as compared with vehicle-treated cells. These data demonstrate transcriptional suppression as a molecular explanation for glucocorticoid-induced down-regulation of beta 1-adrenergic receptors.

Comparison of the concentration of messenger RNA encoding four muscarinic receptor subtypes in control and Alzheimer brains

We determined the concentration of the messenger RNA species which encode four (m1–m4) of the five cloned muscarinic receptors in brains of Alzheimer's disease patients as compared to age-matched controls. Assays were performed using the quantitative method of DNA-excess solution hybridization in the cerebral cortex (frontal, temporal and occipital), hippocampus, nucleus basalis of Meynert and brainstem. The results suggest a statistically significant decrease in the m1 muscarinic receptor message in the temporal and occipital cortex, with no change in other regions. There was no change in the level of mRNA encoding the m2, m3 or m4 receptors in any of the brain regions studied.

Comparison of the level of mRNA encoding m1 and m2 muscarinic receptors in brains of young and aged rats

We compared the concentration of mRNA encoding the m1 and m2 muscarinic receptors in several brain regions obtained from young (5–8 months) and aged (24–28 months) male Fischer 344 rats. DNA-excess solution hybridization was employed as a quantitative measure of mRNA concentration. The results indicate the absence of changes in the m1 receptor message with aging in the cerebral cortex, hippocampus and striatum. While there was no statistically significant aging-associated alteration in the concentration of the message encoding the m2 receptor in the thalamus, midbrain, cerebellum and brainstem, there was a decrease in the message level in the hypothalamus.

Selective regulation of beta 2-adrenergic receptor gene expression by interleukin-1 in cultured human lung tumor cells.

The regulation of beta 1- and beta 2-adrenergic receptors (beta 1AR and beta 2AR) and receptor gene expression by interleukin-1 alpha (IL-1 alpha) was studied in cultured A549 human lung adenocarcinoma cells. The density and affinity of beta 1 AR and beta 2 AR were analyzed by computerized curve fitting of 125I-pindolol binding and its displacement by subtype selective antagonists. Steady state levels of receptor mRNAs were quantified by DNA excess solution hybridization assays. A549 cells in preconfluent cultures had fewer beta 1AR than beta 2AR (beta 1: 1.9 +/- 0.3 vs beta 2: 4.0 +/- 0.5 fmol/mg protein, means +/- SE), but lost most of their beta 2 AR upon reaching confluency (beta 1: 2.7 +/- 0.4, beta 2: 0.8 +/- 0.3 fmol/mg). Incubation of preconfluent cells for 24 hr with 20 pM of human recombinant IL-1 alpha did not modify the density of either of the beta AR subtypes. Similar incubations of confluent cells increased the density of beta 2 AR from 0.8 +/- 0.3 to 4.2 +/- 0.9 fmol/mg, while the density of beta 1 AR and the antagonist affinities of both receptors remained unaltered. The IL-1 alpha-induced increase in beta 2 AR density in confluent cells was antagonized in a concentration-dependent manner by a recombinant protein antagonist of type I IL-1 receptors (IC50: 0.2 nM). The IL-1 alpha-induced increase in beta 2AR density was preceded by an increase in the steady state level of beta 2AR mRNA, while levels of beta 1AR mRNA remained unchanged. IL-1 alpha increased the stability as well as the rate of transcription of beta 2AR mRNA. These findings demonstrate for the first time that activation of type I IL-1 receptors in A549 cells leads to a cell density-dependent, selective upregulation of beta 2AR, and that the mechanism of this effect involves increased formation and stability of the beta 2AR message.

Thyroid hormones transcriptionally regulate the beta 1-adrenergic receptor gene in cultured ventricular myocytes

Exposure of ventricular myocytes in primary culture to triiodothyronine (T3) increased the number of beta 1-adrenergic receptors per cell by 2.1 +/- 0.3-fold (n = 7) within 48 h. Immunoblots of membranes prepared from myocytes revealed a marked increase by T3 in the 64-kDa species of the beta 1-adrenergic receptor. Steady-state levels of beta 1- and beta 2-adrenergic receptor mRNAs quantified by DNA-excess solution hybridization were 0.26 +/- 0.06 and 0.81 +/- 0.05 amol of beta-adrenergic receptor mRNA/micrograms of total cellular RNA, respectively (n = 4). beta 1-Adrenergic receptor mRNA increased to 0.90 +/- 0.07 amol/micrograms RNA by 2-4 h after exposure to T3 and then declined by 6 h to twice that of control cells. beta 2-Adrenergic receptor mRNA levels were unaffected by T3. Northern blot hybridization also showed a rapid and sustained increase of 2.2 +/- 0.4-fold (n = 4) in beta 1-adrenergic receptor mRNA. The rate of beta 1-adrenergic receptor gene transcription assessed by nuclear run-on transcription assays increased by 3.4 +/- 0.4-fold in cells treated for 30 min with T3. Ventricular cells contained nuclear T3 receptors and mRNAs of c-erbA genes that encode T3 binding proteins. These studies indicate that thyroid hormones regulate the cardiac beta-adrenergic receptor-adenylate cyclase system by controlling the rate of transcription of the beta 1-adrenergic receptor gene. This regulation involves nuclear T3 receptors and appears to be exerted in a tissue-specific manner.

DFP-induced regulation of cardiac muscarinic receptor mRNA in vivo measured by DNA-excess solution hybridization

Relationship between in vivo down-regulation of cardiac muscarinic receptors and changes in their encoding mRNA was investigated. Rats were treated either once or for ten days with an irreversible inhibitor of acetylcholinesterase, followed by measurements of cardiac acetylcholinesterase, the density and affinity of muscarinic receptors, and the concentration of mRNA coding for these receptors. mRNA was quantitated using the sensitive method of DNA-excess solution hybridization. Our data indicate that while short-term treatment resulted in a marked decrease in the density of cardiac muscarinic receptors by 34%, there was no accompanying significant change in the concentration of their mRNA. In contrast, long-term inhibition of acetylcholinesterase significantly decreased the concentration of both receptors and mRNA by 40% and 29%, respectively. These results are indicative of multiple mechanisms of down-regulation of cardiac muscarinic receptors, some of which might involve alterations at the transcriptional level.

Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2.

The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.

G-protein subunit mRNA levels in rat heart, liver, and adipose tissues: Analysis by DNA-excess solution hybridization

The steady-state levels of mRNAs for the G-proteins G iα2, G oα, and the G β-subunits common to each were established in rat adipose, heart and liver. Uniformly-radiolabeled, single-stranded antisense probes were constructed from cDNAs or assembled from oligonucleotides. Direct comparison of the steady-state levels of the G-protein mRNAs was performed under identical assay conditions, and on a molar basis. In adipose, liver and heart, G sα mRNA was more abundant than mRNA for G oα, G iα, and G β. In adipose tissue, mRNA levels were as follows: 19.4, 7.6, 7.0, and 2.3 amol mRNA per μg total cellular RNA for G sα, G β, G iα2, and G oα, respectively. In heart G sα mRNA was less abundant than in adipose, but the relative trend among the G-protein subunits was the same. In liver, G β mRNA was more abundant than either G oα or G iα2. G oα mRNA levels ranged from 1.2 to 2.3 amol/μg total RNA in liver and adipose, respectively. The present work demonstrates the many advantages of this strategy when applied to the study of a family of homologous, low-abundance proteins and establishes for the first time the molar levels of G iα2, G sα, G oα, and G β-subunit mRNAs in several mammalian tissues.

Beta 1- and beta 2-adrenergic receptor expression in differentiating 3T3-L1 cells. Independent regulation at the level of mRNA.

Regulation of two highly homologous GTP-binding regulatory protein- (G-protein) linked receptors, beta 1- and beta 2-adrenergic receptors, was probed at the level of mRNA in differentiating 3T3-L1 cells. Expression of the two receptor subtypes at the protein level was defined by competition of radioligand binding with CGP-20712A, a highly selective beta 1-adrenergic antagonist. 3T3-L1 fibroblasts express equivalent levels of beta 1- and beta 2-adrenergic receptors. Following treatment with dexamethasone and isobutylmethyl xanthine (IBMX), 3T3-L1 cells differentiate to adipocytes and express 4-fold more receptor, predominantly beta 2-subtype (beta 1-/beta 2- ratio, 5:95). Regulation of beta 1- and beta 2-receptor mRNA levels by differentiation, as well as by steroid alone and IBMX alone was probed by DNA excess solution hybridization. A beta 1-receptor antisense probe was constructed from double-stranded DNA assembled from synthetic oligonucleotides. In untreated 3T3-L1 fibroblasts the steady-state levels of beta 1- and beta 2-adrenergic receptor mRNA were equivalent (approximately 1.2 amol mRNA/micrograms total cellular RNA). beta 2-Adrenergic receptor mRNA levels increased 3-fold as 3T3-L1 fibroblasts were differentiated to adiopcytes (day 7). mRNA levels for beta 1-adrenergic receptor, in contrast, increased at day 2, but thereafter declined, falling to less than 0.05 amol mRNA/micrograms total cellular RNA by day 7 in adipocytes. A 7-day challenge with dexamethasone reduced by 50% beta 1-adrenergic receptor mRNA levels. Treatment with IBMX alone reduced mRNA levels for both receptor subtypes. Neither steroid nor IBMX alone promoted differentiation. The present work, for the first time, demonstrates (i) the mRNA levels on a molar basis for two highly homologous G-protein-linked receptors expressed in a single cell, (ii) independent regulation of their mRNA levels that correlates well with receptor expression, and (iii), that it is differentiation in 3T3-L1 cells per se and not treatment with glucocorticoid or IBMX alone that promotes the up-regulation of the beta 2-receptor transcripts and down-regulation of beta 1-receptor transcripts.

G-Protein mRNA levels during adipocyte differentiation

G-protein-mediated transmembrane signaling in 3T3-L1 cells is modulated by differentiation. The regulation of G-protein expression in differentiating 3T3-L1 cells was probed at the level of mRNA by DNA-excess solution hybridization. Pertussis toxin-catalyzed ADP-ribosylation of G-protein α-subunits increased as fibroblasts differentiate to adipocytes. Steady-state levels of mRNA for G iα2 and G oα, in contrast, declined sharply. Immunoblotting with antipeptide antibodies specific for G iα2, too, revealed a decline in the steady-state expression of this pertussis toxin substrate. ADP-ribosylation of G sα by cholera toxin was less in the adipocyte than fibroblast. Analysis by immunoblotting revealed only a modest decline in G sα. Analysis of mRNA levels also demonstrated a decline for G sα. mRNA levels for the G β-subunits rose initially (25%) on day 1, declined from day 1 to day 3, and remained 25% lower in adipocytes than in fibroblasts. In 3T3-L1 adipocytes the molar amounts of subunit mRNAs were: 60.6 (G sα); 2.1 (G iα2); and 1.5 (G oα) amol/μg total cellular RNA. In rat fat cells these mRNA levels were 19.4 (G sα); 7.0 (G iα2); and 2.3 (G oα). These data demonstrate that for G iα2 and G oα alike mRNA and protein expression decrease, not increase, in differentiation. A substrate for pertussis toxin other than G iα2 and G oα appears to be responsible for the increase in toxin-catalyzed labeling that accompanies differentiation of 3T3-L1 cells.

Agonist-induced Destabilization of β-Adrenergic Receptor mRNA: Attenuation of glucocorticoid-induced up-regulation of β-adrenergic receptors

β-Adrenergic receptor expression and receptor mRNA levels are down-regulated by β-adrenergic agonists and up-regulated by glucocorticoids. The interaction between these two opposing regulatory pathways was investigated at the levels of receptor and receptor mRNA in DDT1 MF-2 hamster vas deferens cells. Dexamethasone blunted a marked decrease in receptor expression induced by isoproterenol alone, as made visible by indirect immunofluorescence using antireceptor antibodies. Receptor mRNA levels were quantified by DNA-excess solution hybridization. Dexamethasone stimulated a sharp increase in receptor mRNA at 4 h following the addition of steroid in either the absence or the presence of isoproterenol. By 12 h, dexamethasone treatment resulted in a new steady-state level of receptor mRNA double that observed in untreated cells. Isoproterenol blunted the dexamethasone effect observed at 12 h. Cells treated with isoproterenol and dexamethasone in combination displayed a new steady-state level only 30% greater than untreated cells. Measured by nuclear run-on assays, transcription rates of the receptor gene were unaffected in cells challenged with isoproterenol alone. Dexamethasone, in contrast, stimulated a 4-fold increase in β2-adrenergic receptor gene transcription. Isoproterenol and dexamethasone in combination promoted a transcription rate comparable to dexamethasone alone. The half-life of receptor mRNA in untreated and dexamethasone-treated cells was 12 h. In contrast, β-adrenergic receptor mRNA half-life declined to 5 h in cells that were treated with isoproterenol in the presence or absence of dexamethasone. Agonist-promoted destabilization and steroid-induced transcription provide mechanisms for the interplay of two opposing pathways controlling receptor mRNA levels.

Differential effects of dietary fat on the tissue-specific expression of the apolipoprotein A-I gene: relationship to plasma concentration of high density lipoproteins.

Isocaloric substitution of polyunsaturated fat for saturated fat reduces concentrations of total plasma cholesterol and high density lipoproteins (HDL) in nonhuman primates. The biochemical mechanisms through which polyunsaturated fat lowers plasma HDL concentrations are not well understood but must involve changes in HDL production or HDL clearance from plasma, or both. To determine whether dietary polyunsaturated fat (P/S = 2.2) alters apolipoprotein (apo) A-I production, African green monkeys (Cercopithecus aethiops) were fed diets containing polyunsaturated fat or saturated fat (P/S = 0.3) each in combination with high (0.8 mg/kcal) and low (0.03 mg/kcal) amounts of dietary cholesterol. Animals fed polyunsaturated fat at either cholesterol level had lower plasma concentrations of total cholesterol and HDL cholesterol. Plasma apoA-I concentration was reduced by 16% by polyunsaturated fat in the high cholesterol group. The rate of hepatic apoA-I secretion, as estimated by the accumulation of perfusate apoA-I during recirculating liver perfusion, was reduced by 19% in animals consuming the high cholesterol, polyunsaturated fat diet. Hepatic apoA-I mRNA concentrations, as measured by DNA-excess solution hybridization, also were reduced by 22% in the high cholesterol, polyunsaturated fat-fed animals. In contrast, intestinal apoA-I mRNA concentrations were not altered by the type of dietary fat. Plasma apoA-II and hepatic apoA-II mRNA concentrations also were not altered by the type of dietary fat. These data indicate that dietary polyunsaturated fat can selectively alter the expression of the apoA-I gene in a tissue-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid Hormones Regulate G-protein β-Subunit mRNA Expression in Vivo

Abstract Thyroid hormones exert effects on the hormone-sensitive adenylate cyclase. Regulation of the expression of Gi (Gi alpha 2) and Gs by thyroid hormones in vivo was investigated at the level of mRNA. Steady-state levels of the mRNA for Gi alpha 2 and Gs alpha, as well as the G beta-subunits, were quantified using DNA excess solution hybridization analysis. Regulation of protein and mRNA expression in adipose tissue was investigated in hypothyroid, euthyroid, and hyperthyroid rats. In euthyroid animals, steady-state levels of mRNA (amol/microgram RNA) were 13.8, 5.9, and 5.7 for Gs alpha, Gi alpha 2, and G beta 1,2, respectively. Activation of adenylate cyclase by Gs is unaffected by thyroid status. Both Gs alpha and Gs alpha mRNA levels in hypothyroid rats were the same as those of controls (euthyroid). The inhibitory control of adenylate cyclase, in contrast, is markedly potentiated in hypothyroid rats. The expression of G1 alpha s and G beta-subunits was increased in hypothyroidism. Whereas Gi alpha 2 mRNA levels remained essentially unchanged, G beta 1,2 mRNA levels were observed to increase 45% in the hypothyroid state. In the hyperthyroid state G beta 1,2 mRNA levels were observed to decline by 35%. Regulation of G-protein subunit expression, at the level of mRNA, appears to be one component of permissive hormone action on transmembrane signalling.

Agonist Regulation of β-Adrenergic Receptor mRNA: Analysis in S49 Mouse Lymphoma Mutants

Abstract Agonist-promoted down-regulation of beta-adrenergic receptor mRNA was investigated in S49 mouse lymphoma variants with mutations in elements of hormone-sensitive adenylate cyclase. In wild-type cells steady-state levels of beta-adrenergic receptor mRNA were established by DNA-excess solution hybridization to be 1.72 +/- 0.08 (n = 8) amol/microgram total cellular RNA. Receptor mRNA levels declined 35-45% in response to stimulation by the beta-adrenergic agonist (-)isoproterenol or forskolin as described previously in DDT1 MF-2 cells (Hadcock, J. R., and Malbon, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5021-5025). Agonist-promoted cAMP accumulation and down-regulation of receptor mRNA were analyzed in three variants with mutations in Gs alpha (H21a, unc, cyc-) and a single variant lacking cAMP-dependent protein kinase activity (kin-). H21a (Gs alpha coupled to receptor, but not to adenylate cyclase), unc (Gs alpha uncoupled from receptor), and cyc- (lacking Gs alpha) variants accumulated cAMP and down-regulated beta AR mRNA in response to forskolin. In unc and cyc- cells isoproterenol failed to stimulate cAMP; accumulation and down-regulation of receptor mRNA was not observed. H21a cells, in contrast, displayed agonist-promoted regulation of beta-adrenergic receptor mRNA but only basal levels of cAMP accumulation in response to isoproterenol. The kin- cells displayed cAMP accumulation in response to forskolin as well as to isoproterenol but no down-regulation of receptor mRNA or receptor expression. Taken together these data demonstrate several features of agonist-promoted down-regulation of mRNA: (i) cAMP-dependent protein kinase activity is required for down-regulation of mRNA (kin-), although elevated cAMP accumulation is not (H21a); (ii) functional receptor-Gs coupling is required (H21a), and clones lacking Gs alpha (cyc-) or receptor Gs coupling (unc) lack the capacity to down-regulate mRNA in response to agonist; and (iii) in the presence of basal levels of cAMP and cAMP-dependent protein kinase activity, functional receptor-Gs coupling (H21a) to some other effector other than adenylate cyclase may be propagating the signal.

Physiological regulation at the level of mRNA: analysis of steady-state levels of specific mRNAs by DNA-excess solution hybridization.

Recent appreciation for how systems may be regulated at the protein and mRNA levels has dictated that physiological questions be addressed at both. The application of novel and sensitive biochemical and molecular biological techniques may be necessary to answer questions fundamental to our understanding of physiological regulation. For measurement of low-abundance proteins, such as receptors for hormones and growth factors, and for measurement of their corresponding mRNAs, the need for highly sensitive assays is critical. The present work focuses both on recent studies of the regulation of steady-state levels of mRNAs and on how mRNAs are quantified by DNA-excess solution hybridization assays using highly specific single-stranded probes. Examples of physiological regulation are provided in which analysis of mRNA levels has been a key facet to our understanding of the mode of regulation. The construction and characterization of probes for solution hybridization assays are described using the hamster beta 2-adrenergic receptor as a model. Studies on the regulation of beta-adrenergic receptors by agonists and by permissive hormones as well as investigations on the regulation of apolipoproteins by estrogen and by cholesterol in vivo are used to illustrate the many advantages of analyzing the steady-state levels of specific mRNAs by DNA-excess solution hybridization.

Quantification and regulation of apolipoprotein E expression in rat Kupffer cells.

Apolipoprotein E (apoE) is synthesized by a wide variety of cells including cells of the monocyte-macrophage lineage. In order to assess the quantitative significance of apoE synthesis in a mature tissue macrophage, apoE synthesis was compared in Kupffer cells and hepatocytes isolated from rat liver. Immunoreactive apoE synthesized by both cell types exhibited identical isoform patterns when examined by high-resolution two-dimensional gel analysis. ApoE synthesis was not detected in hepatic endothelial cells. Northern blot analysis using a rat apoE cDNA probe demonstrated a single mRNA species of approximately 1200 nucleotides in freshly isolated hepatocytes and Kupffer cells. The absolute content of apoE mRNA in each cell type was determined with a DNA-excess solution hybridization assay. The apoE mRNA content (pg/microgram RNA) for Kupffer cells and hepatocytes was 35.7 and 98.8, respectively. Accounting for cellular RNA content and the population size of each cell type in the liver, Kupffer cells were calculated to contain about 0.7% of liver apoE mRNA; hepatocytes account almost quantitatively for the remainder. These results suggest that Kupffer cells are not major contributors to the plasma apoE pool. After intravenous injection of bacterial endotoxin, apoE mRNA was decreased in freshly isolated Kupffer cells whereas whole liver showed no change in apoE mRNA. Endotoxin treatment had no effect on the apoE mRNA content in several peripheral tissues. These results indicate that apoE expression in vivo is differentially regulated by endotoxin in Kupffer cells as compared to hepatocytes or apoE-producing cells in peripheral tissues.

Kinetics of interleukin 2 mRNA and protein produced in the human T-cell line Jurkat and effect of cyclosporin A.

The kinetics of interleukin 2 mRNA accumulation in the leukemic T-cell line Jurkat, which can be induced with phytohemagglutinin and phorbol 12-myristate 13-acetate to produce large amounts of interleukin 2, was analyzed by a modified DNA-excess solution hybridization assay using a 5'-32P-labeled oligodeoxyribonucleotide 30 bases long as probe. Cyclosporin A was used as a valuable tool to gain more insight into the quantitative aspects of interleukin 2 production, on the basis of the assumption that transcription of the interleukin 2 gene is completely inhibited shortly after administration of cyclosporin A. The half-life of interleukin 2 mRNA was estimated to be approximately 2 h. With the aid of simple mathematical models, we have been able to relate the concentration of interleukin 2 protein in the supernatant to the interleukin 2 mRNA kinetics. This novel quantitative kinetic analysis revealed that, independent of the absence or presence of cyclosporin A, interleukin 2 protein is synthesized at a rate of approximately 1.3 molecules per molecule of interleukin 2 mRNA per second and secreted within 2 h after it is synthesized and that its half-life in the supernatant is approximately 10 h.

Regulation of beta-adrenergic receptors by "permissive" hormones: glucocorticoids increase steady-state levels of receptor mRNA.

Incubation of DDT1 MF-2 hamster vas deferens cells with glucocorticoids results in a marked increase in beta-adrenergic receptor (beta AR) number. The increase in receptor number was visualized by indirect immunofluorescence with antiserum specific for the beta AR and was verified by radioligand binding. The steady-state levels of beta AR mRNA were quantified in untreated (control) and glucocorticoid-treated cells by DNA-excess solution hybridization using a single-stranded probe corresponding to nucleotides +12 to 182 of the hamster beta 2AR cDNA coding region. The steady-state level increased from 0.37 pg of beta AR mRNA per microgram of total cellular RNA in untreated cells to 1.05 pg of beta AR mRNA per microgram of RNA in cells treated with dexamethasone (500 nM) for 2-4 hr. After this sharp transient peak, the steady-state level of receptor mRNA declined by 6 hr to a level approximately twice that of the untreated cells. Half-maximal effects were achieved at 20-40 nM dexamethasone. Testosterone (500 nM) and 17 beta-estradiol (500 nM), in contrast, did not alter the steady-state levels of beta AR mRNA. Actinomycin D, a potent inhibitor of transcription, abolished the dexamethasone-induced increase in beta AR mRNA, suggesting that the permissive hormone effect was exerted on gene transcription. The half-life of the receptor mRNA measured in the presence of actinomycin D was found to be 12 hr in both the untreated and the dexamethasone-treated cells. These studies provide a molecular explanation for the well-known regulation of GTP-binding protein (G-protein)-linked cell-surface receptors by permissive hormones.

Down-regulation of beta-adrenergic receptors: agonist-induced reduction in receptor mRNA levels.

Incubation of DDT1 MF-2 hamster vas deferens cells with beta-adrenergic agonists results in a time- and concentration-dependent decreases in both beta-adrenergic receptor (beta AR) responsiveness and receptor number. Receptor mRNA levels were quantified by DNA-excess solution hybridization by using a 170-nucleotide single-stranded probe derived from the hamster beta 2AR cDNA. RNA blot analysis of poly(A)+-selected RNA with the solution probe revealed a 2.2-kilobase species. Digestion of the RNA/solution probe mixture with S1 endonuclease revealed a single species of RNA (170 bases) that was protected by the solution probe. DDT1 MF-2 cells were found to contain 0.38 pg of beta AR mRNA per microgram of total cellular RNA. Incubation (16 hr) with isoproterenol decreased beta AR mRNA levels in cells by 40%. This agonist-induced decrease in receptor mRNA levels was found to be dependent on the time of incubation and the dose of agonist. The decrease in beta AR mRNA was half-maximal at 0.1-0.5 microM isoproterenol. The beta-adrenergic antagonists CGP 20712A (beta 1-selective) and ICI 118,551 (beta 2-selective) blocked in a dose-dependent fashion the ability of isoproterenol to effect receptor mRNA levels. The beta 2-adrenergic antagonist displayed a potency 25-fold greater than that of the beta 1-adrenergic antagonist, in agreement with the subtype of receptor (beta 2) expressed by these cells. For down-regulated cells in which receptor mRNA levels declined in response to agonist, the addition of the antagonist ligand (-)-propranolol (1 microM) was able to restore receptor mRNA levels to 90% of the control value within 12 hr. Full recovery of steady-state beta AR mRNA was achieved within 60 hr. These studies provide a molecular explanation for the down-regulation of GTP-binding regulatory protein (G protein)-linked cell-surface receptors that accompanies desensitization.