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The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on growth factor-coupled activation of nuclear protein kinase C (nPKC) and on the subcellular distribution of PKC activity in rat splenocytes were investigated. Seven days after a single injection of TCDD (50 micrograms/kg body weight), cytosolic and particulate PKC activity was significantly higher in splenocytes from TCDD-treated rats or pair-fed control rats compared to ad libitum-fed animals. In a separate experiment, purified splenocyte nuclei from TCDD-treated animals and controls were used to study activation of nPKC by growth factors and other trophic agents. Growth factor-stimulated nPKC activation was attenuated in splenic nuclei from TCDD-treated rats compared to vehicle-treated controls. Evidence presented here suggests that the cellular mechanism of TCDD toxicity leading to immunosuppression in rodents may be mediated in part by uncoupling of growth factor receptors linked to PKC activation at the level of the nucleus. However, changes in total splenocyte PKC activity appear to be correlated with hypophagia since cytosolic and particulate PKC levels were elevated in TCDD-treated rats and their pair-fed partners.

The adenohypophyseal polypeptide hormone prolactin is a potent liver mitogen, stimulating cell cycle progression, an effect that appears coupled to increasing protein kinase C activity in membrane and nuclear fractions. Here, we examine whether hepatocyte proliferation, stimulated by partial hepatectomy, is associated with altered serum prolactin or protein kinase C activation. Within 5-15 min of liver resection, serum prolactin concentrations elevate significantly. Protein kinase C activity in hepatic cytosol decreases significantly, and membrane and nuclear PKC activity increase by 30 min. Hypophysectomy prior to partial hepatectomy abrogates any effect of liver resection on protein kinase C activation in the hepatic remnant. Based upon these data, it is suggested that the rapid increase in serum prolactin seen after partial hepatectomy may be linked to protein kinase C activation, which in turn stimulates the hepatic proliferative response that is essential for hepatic regeneration.

Prolactin (PRL) and other trophic factors rapidly activate a nuclear pool(s) of protein kinase C (nPKC) in purified splenocyte nuclei. The PRL also enhanced [2-3H]glycerol incorporation into nuclear mono- and triacylglycerol. An assay was devised which not only probed the ability of the hormone to activate protein kinase C (PKC) but also demonstrated the presence of nuclear substrates. Using this methodology, a biphasic concentration-response curve to PRL was observed. Heterologous species of PRL and various growth factors also activated nPKC. The PRL-induced nPKC stimulation was antagonized by various immunomodulators, G protein-coupling inhibitors, PKC inhibitors, a calmodulin inhibitor, and a peripheral benzodiazepine agonist and antagonist. A monoclonal antibody to PKC, anti-rat PRL antiserum and a monoclonal anti-rat PRL receptor antibody antagonized PRL-induced PKC-dependent nuclear phosphorylation, further implicating nPKC and a PRL receptor-mediated activation process. Nuclear PKC may be a major target for trophic regulation in response to both positive and negative growth signals.

We have examined the actions of vasoactive intestinal peptide (VIP) and certain other known immune modulators on a nuclear pool(s) of protein kinase C (PKC) in isolated rat splenocyte nuclei. Rat splenocyte nuclei pure by enzymatic and electron microscope criteria demonstrated a time- and concentration-dependent activation of nuclear PKC (nPKC) by VIP. A biphasic pattern of three bell-shaped curves was observed with peak phosphorylation at 10(-15), 10(-9) and 10(-6)M VIP. The phosphorylation of endogenous nuclear substrates was characterized as a PKC-mediated event by use of three known PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), sphingosine, and staurosporine, which produced similar phosphate incorporation measurements. Also, this activity was blocked with the addition of a monoclonal antibody to PKC. Inhibitors of the ability of VIP to activate nPKC included somatostatin, 8-bromo-cAMP, peripheral benzodiazepine receptor modulators, and the PKC inhibitors, sphingosine and staurosporine. These data have direct relevance to our knowledge of cell-mediated immunity.

Nuclear pool(s) of protein kinase C (PKC) may be a common target for hormones and growth factors which affect the trophic state of cells. The data presented demonstrate a time and dose-dependent activation of nuclear PKC by the HIV coat protein, gp120, in isolated nuclei from rat spleen and hippocampus. This gp120-stimulated PKC response was blocked by specific PKC inhibitors, a monoclonal antibody to PKC, and a monoclonal antibody directed against the murine T4 analog, L3T4. It is suggested that the gp120 interaction with the nuclear trophic factor-PKC system may impair normal gene expression, and thus result in the clinical symptoms associated in AIDS infection.

The stimulatory effects of adenosine analogues on a nuclear pool of protein kinase C (PKC) were examined using isolated rat splenocyte nuclei. Nuclear receptors met pharmacological criteria of A1 adenosine receptors including a potency profile in which cyclopentyladenosine (CPA), a selective A1 agonist, was more potent than 2-phenylaminoadenosine (2PAA), a selective A2 agonist. The selective A1 receptor agonist N6-1-(phenyl-2R-propyl) adenosine (R-PIA) activated PKC whereas the S diastereomer did not. The adenosine-induced PKC response could be attenuated using a monoclonal antibody to PKC, an A1 receptor antagonist, three known PKC inhibitors and pertussis toxin (PTX). The results suggest that adenosine may exert immunomodulatory effects through the activation of nuclear PKC.

In the genetic skin disease psoriasis, the number of Proliferating cells is 12-fold greater in lesional (involved) and 2.5-fold greater in uninvolved epidermis than in normal epidermis. Since increased polyamine biosynthesis is associated with Cell proliferation, we measured polyamine levels and the activities of the polyamine biosynthetic enzymes from biopsies of involved and uninvolved psoriatic epidermis and normal epidermis. Polyamine (putrescine, spermidine, and spermine) levels were: (1) higher in involved than in uninvolved epidermis (N = 7, P < 0.05); (2) higher in uninvolved (N = 7) than in normal (N = 8) epidermis (P < 0.02); (3) higher in urine from psoriatic patients (N = 7) than in normal (N = 8) urine (P < 0.02). The activities of ornithine decarboxylase, and both putrescine and spermidine-stimulated S-adenosyl-L-methionine decarboxylase, were 6- fold higher in involved versus uninvolved (N = 12) or normal epidermis (N = 12, P < 0.01). After 24 hr of glucocorticoid pretreatment of lesions the activities of all 3 enzymes were markedly inhibited (N = 12, P < 0.02). Methylglyoxal bis (guanylhydrazone) and α-methylor-nthine inhibited the in vitro activities of S-adenosyl-L-methionine decarboxylase and ornithine decarboxylase, respectively, from lesional psoriatic epidermis. In conclusion: (1) the number of proliferating cells in the hyperproliferative epidermis of psoriasis and the increase polyamines are correlated; (2) glucocorticoid inhibits lesional polyamine biosynthetic enzyme activity and is known to clear psoriatic lesions; (3) non-glucocorticoid inhibitors of polyamine formation such as those given above might improve psoriatic lesions; (4) the discovery of elevated polyamine biosynthesis in both uninvolved and involved epidermis may lead to a better understanding of misregulated cell proliferation in this disease.

Ornithine decarboxylase, the initial enzyme in the polyamine biosynthetic pathway, is induced in target tissues in response to a variety of trophic agents including polypeptide and amine trophic hormones, cyclic AMP analogs, drugs, and trophic steroid hormones. The induction of ornithine decarboxylase in these systems is regulated at a transcriptional level and is proportional to the extent of stimulation. Because of its rapid half-life (10-20 min), a general maximum of induction is detectable within 4-5 h of stimulation, and its induction pattern can serve as a rapid, specific index of increased RNA and protein synthesis. Implications for its usefulness to pharmacologists, endocrinologists, physiologists, and biochemists are summarized.

Prolactin receptors have been identified for the first time on human peripheral blood lymphocytes. These receptors are present on T- and B-cells as well as monocytes. The specific binding of [ 125 I]prolactin to these cells can be selectively enhanced at certain concentrations and blocked by higher concentrations of cyclosporine, a known immunosuppressive agent which inhibits the mitogenesis of T-cells. Prolactin also induces ornithine decarboxylase, a key growth regulatory enzyme, in lymphocytes. Therefore, we suggest that the lymphocyte prolactin receptor may be involved in regulating lymphocyte function, and that one of the actions of cyclosporine is to block this rather ubiquitously occurring receptor.

[3H]Ro 5-4864 binds to Nb 2 node lymphoma cells in a specific saturable and reversible fashion. Scatchard analysis of specific binding data reveals a single, homogeneous class of whole cell binding sites with a Kd of 3.94 +/- 0.22 nM and a Bmax value of 155 +/- 11 fmol (Ro 5-4864 bound)/2 x 10(6) cells. Ro 5-4864, a reported peripheral benzodiazepine receptor agonist both inhibits (10(-6) M) and potentiates (10(-9) M) the mitogenic action of prolactin on the Nb 2 node lymphoma cells. Interestingly, PK 11195, an antagonist, potentiates (10(-9) M) the mitogenic activity of prolactin in these cells. The actions of both Ro 5-4864 and PK 11195 seem to be mediated through a common receptor type since a 10(-6) M concentration of either agent will block the others potentiating action. Furthermore, the simultaneous addition of a 10(-9) M concentration of Ro 5-4864 and PK 11195 does not further increase the effect on prolactin stimulated mitogenesis. Clonazepam, a central benzodiazepine receptor agonist has no effect on prolactin-stimulated mitogenesis in this system. These data suggest that the Nb 2 node lymphoma cells possess a peripheral-type benzodiazepine receptor. In these cells, this receptor seems to serve the function of modulating the ability of the growth factor, prolactin to initiate the mitogenic process. These studies also suggest that Ro 5-4864 is functioning as a partial agonist rather than a 'pure' agonist for the peripheral benzodiazepine receptor in this system.

The goitrogen, propylthiouracil (PTU), when administered in the diet of white leghorn chickens resulted in a marked increase in weight of the thyroid gland within 7 days which was maximal (160% of control) within 14 days. This system was used to determine the alterations in cyclic AMP-dependent protein kinase activity ratios and to assess the amounts of type I and type II cyclic AMP-dependent protein kinases present in the thyroid as well as any changes related to hypertrophy and hyperplasia of the thyroid. There was a marked elevation in the cyclic AMP-dependent protein kinase activity ratio ( −cAMP +cAMP ) 7 days after initiation of treatment as well as an increase in the total amount of supernatant cyclic AMP-dependent protein kinase activity (+cAMP) which doubled within 14 days of dietary PTU. Both type I and type II cyclic AMP-dependent protein kinase activities were detectable in the thyroid and adenohypophysis. Type I comprised 10% and type II 90% of the total cyclic AMP-dependent protein kinase activity in both tissues. The total amount of type I protein kinase activity increased significantly by 7 days. By 14 days, both type I and II protein kinases were increased twofold. The amount of type I returned to a control value by 21 days whereas type II remained elevated. Adenohypophysial type II protein kinase activity decreased to 70% of control by 14 days. Cyclic AMP is known to exert its effect on trophic responses through the activation of cyclic AMP-dependent protein kinase. These data implicate both activation of the enzymes as well as increased amounts of types I and II protein kinase activity in the thyroid in response to goitrogen-induced hypertrophy and hyperplasia.

Creatine kinase (CK) activity and isozyme patterns were assessed in newborn and adult rat anterior tibial muscle in response to denervation. Total CK activity was low in the control neonatal muscle, gradually increasing to the adult level within 1 month. Denervation prevented this normal increase, and, therefore, CK activity was reduced to 25% of control at 2 months. In the denervated adult muscle, total CK activity decreased to 50% of control within 3 weeks and remained at that level. Denervation of neonatal muscle resulted in a greater conservation of MB isozyme compared with controls. The alteration in BB isozyme expression was even more dramatic with a 33-fold difference expressed at 2 months in terms of percent total CK in denervated vs. control muscle. In denervated adult muscle, MB and BB isozyme activities increased gradually, attaining levels 3-fold and 13-fold, respectively, above control muscle at the end of the experimental period.

The effects of insulin and glucagon on CAMP accumulation, protein kinase activation, and glycogenolysis were investigated in isolated rat hepatocytes. Glucagon (0.01 nM to 10 μM) increased the activation state of protein kinase and the rate of glucose accumulation. Addition of 1.0 nM insulin to cells preincubated with 0.1 nM glucagon attenuated the rate of glucose accumulation, but did not alter the protein kinase activity ratio. Addition of 0.1 nM glucagon to cells pre-incubated with 1.0 nM insulin caused a rapid activation of protein kinase; however, glycogenolysis was not immediately affected. These effects were enhanced with pharmacological concentrations of glucagon and insulin. These data indicate that the degree of protein kinase activation does not always correlate temporally or quantitatively with rates of glycogenolysis in liver cells exposed to insulin and glucagon.

Ornithine decarboxylase, an important enzyme in growth regulation, is increased in CHO cells in G1 phase of the cell cycle and decreases as the cells progress into S phase. S-adenosyl-L-methionine decarboxylase activity, which is dependent on either the presence of putrescine or spermidine for the synthesis of spermidine and spermine respectively, shows a maximal increase in late G1/early S phase which corresponds very closely with the cell cycle phase specific accumulation of spermidine and spermine during S phase. Total culture evaluation of spermidine and spermine, which included extracellular as well as intracellular concentrations, indicated that extracellular accumulations of these polyamines occurred only in G1 and that entry into S phase was concomitant with intracellular accumulation patterns. Hyperthermia (43°C for 1 hour) in mid-G1 phase of the cell cycle resulted in rapid decreases in the activities of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase. In these cells, DNA replication was also not detectable until nine hours after mitosis, a time at which there had been recovery of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase activities. Previous data have further indicated a requirement for polyamine reaccumulation before control DNA replication rates are resumed. We therefore suggest that polyamine biosynthesis and intracellular accumulation are both temporal and quantitative prerequisites for transition through S phase.

A prospective study was carried out over 12 months involving twenty patients with psoriasis vulgaris who were treated with all- trans -aromatic derivative of vitamin A, etretinate. Etretinate was found to be an effective therapy for this skin disorder. Arthritis accompanying psoriasis vulgaris in four of seven of our patients was greatly improved by retinoid therapy. Side effects were found to be dose-related and included mucocutaneous abnormalities as well as abnormalities of blood lipids and liver function tests. Maintenance therapy appears to be required in nearly all patients, with relapse occurring within approximately 8 weeks. Polyamine biosynthesis has been determined previously to be accelerated in patients with psoriasis vulgaris. Polyamines (putrescine [Pu], spermidine [Sp], and spermine [Sm]) were measured in skin samples of six patients with stable plaque-stage psoriasis before treatment and at 4 weeks during treatment. Pu, Sp, Sm levels, as well as the Sp/Sm ratio, fell. Epidermal DNA synthesis is increased in involved and noninvolved psoriatic skin; it was measured before and during treatment (at 4 weeks) in this study and found not to fall significantly during this period of time. Polyamine levels therefore decreased prior to any significant expected decrease in epidermal DNA synthesis measured in both involved and uninvolved skin.

Summary Aminophylline and 1-methyl, 3-isobutylxanthine, inhibitors of phosphodiesterases, caused an increase in the cyclic AMP level and an elevation in the activity of cyclic AMP-dependent protein kinase in the liver of the rat within 10–20 min of administration. The activation of cyclic AMP-dependent protein kinase appeared to be a more accurate measure of cyclic AMP-dependent events than cyclic AMP concentration and was statistically more reliable. After Sephadex chromatography, the protein kinase yielded the same activation pattern as measurable in the crude supernatant of liver.

Creatine kinase (CK), transglutaminase (TGase) and ornithine decarboxylase (ODC), enzymes implicated in the regulation of growth processes, were studied during muscle regeneration subsequent to the injection of bupivacaine into rat tibialis anterior. Within 2 days, the percent BB isozyme of CK detected in the muscle was elevated 70-fold coincident with a marked decrease in total CK activity. The MB isozyme also increased and was 15-fold of control at 4–5 days postinjection. TGase activity was increased significantly to greater than 2-fold of control within 2 days of injection and significantly decreased at days 3 through 7 compared to controls. ODC activity was elevated significantly to 2- to 3-fold of control from 2–7 days after injection. These results suggest an early alteration in the expression of a coordinated battery of genes in this model of muscle degeneration-regeneration. The increased expression of MB and BB isozymes of CK in various human neuromuscular diseases may be a manifestation of an ongoing process of degeneration-regeneration.

Cyclic AMP-dependent protein kinase activation and the induction of ornithine decarboxylase (ODC) were studied along with RNA and DNA synthesis in human peripheral blood lymphocytes following in vitro culture with either mitogens, nonmitogenic agglutinins, or dibutyryl cyclic AMP (DBcAMP). The mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) all resulted in activation of soluble cyclic AMP-dependent protein kinase, the induction of increased levels of ODC activity, and increases in the rate of incorporation of (3H)-thymidine (DNA synthesis) and (3H)-uridine (RNA synthesis) into acid-insoluble material. High concentrations of PHA or Con A, which resulted in maximal cyclic AMP-dependent protein kinase activation, were only slightly mitogenic. The time at which maximum activation of protein kinase occurred differed for each mitogen but, in each case, always preceded ODC induction and increased RNA and DNA synthesis. In Con A-stimulated cultures, cyclic AMP-dependent protein kinase activation was maximal at 4 hr of culture and remained elevated at 24 and 48 hr. In these cultures increased ODC activity was first observed at 6 to 8 hr of culture and preceded increased RNA synthesis which was detectable at 12 hr of culture. DNA synthesis in all mitogen-stimulated cultures was first observed at 48 hr with maximum (3H)-thymidine incorporation being at 72 hr of culture. Cyclic AMP-dependent protein kinase activation, induction of ODC, and increased RNA and DNA synthesis were all shown to be dependent upon lymphocyte-Con A binding and were blocked by early addition of α-methyl mannoside. After 12 hr of culture, α-methyl mannoside addition had little effect on Con A induction of ODC or on RNA and DNA synthesis measured at 24 to 72 hr. In contrast, cyclic AMP-dependent protein kinase activation, measured at 24 hr, could be completely inhibited when α-methyl mannoside was added as late as 12 or 20 hr to Con A-stimulated cultures. The nonmitogenic agglutinins, wheat germ agglutinin (WGA) and Agaricus bisporus lectin (ABL), failed to increase any of the parameters tested. DBcAMP also did not induce ODC or cause any increased RNA or DNA synthesis. However, DBcAMP (10-3 M) did cause activation of cyclic AMP-dependent protein kinase as early as 15 min of culture. DBcAMP activation of protein kinase was concentration-dependent, maximal at 12 hr, and remained elevated through 48 hr of culture. The addition of DBcAMP (10-3 M) to Con A-stimulated cultures caused a 90 to 95% inhibition of ODC activity and RNA and DNA synthesis. This inhibition was concentration-dependent and paralleled the degree of activation of cyclic AMP-dependent protein kinase elicited by each concentration. These results suggest that the selective activation of cyclic AMP-dependent protein kinase and induction of increased ODC activity are early integral events linked to and regulating lymphocyte mitogenesis.

A high-performance liquid chromatography method for the determination of urinary acetyl derivatives of the polyamines putrescine, cadaverine and spermidine is described. This procedure utilizes an ion-exchange column for the separation of acetyl derivatives and the compounds are quantitated by fluorescence after reaction with o-phthalaldehyde. The steps necessary for sample preparation are minimized, and the assay is both sensitive and reproducible. This chromatographic procedure was used for the determination of the urinary acetylated polyamines of seven normal volunteers and three cancer patients.

Within one hr of the administration of cyclosporine to rats, there was a 4-fold elevation in the serum prolactin concentration. Doses of 0.12, 1.2, and 12 micrograms/100 g body weight cyclosporine significantly elevated the serum prolactin level. Higher doses, 120 or 1200 micrograms/100 g body weight cyclosporine resulted in small but insignificant elevations of the serum prolactin concentration. Bromocriptine, a dopamine agonist which inhibits prolactin release from the anterior pituitary, completely blocked the elevation in serum prolactin in response to cyclosporine alone. These data suggest that the ability of cyclosporine to suppress immune function may involve its ability to rapidly produce hyperprolactinemia.

Nuclei and nucleoli isolated from calf liver contain acid-precipitable putrescine, spermidine and spermine conjugates. The polyamines are released upon peptide bond hydrolysis. All of the nuclear putrescine conjugate and a major portion of the polyamine conjugates are localized within the nucleolus. Nuclei and nucleoli also contain, in proportions consistent with the nucleolar/nuclear protein ratio, the putative conjugating enzyme, transglutaminase, as well as amine acceptor substrates to which radiolabeled putrescine can be conjugated by endogenous enzyme. Extraction of the isolated organelles with saline solutions of increasing ionic strength showed a differential distribution of the polyamine derivatives: all the covalently linked putrescine was associated with the less soluble components of the chromatin residue, while the spermidine and spermine conjugates were associated with several salt-extractable protein fractions as well as tightly bound to the chromatin pellet. Mono-gamma-glutamyl putrescine was detected after proteolytic digestion of the 600 mM NaCl fraction, further suggesting the enzymatic action of transglutaminase(s) in the conjugation process.

Children with cystic fibrosis excreted elevated urinary levels of all three polyamines--putrescine, spermidine, and spermine. Heterozygote parents excreted intermediate concentrations of the polyamines, but not levels significantly different from levels in normal controls. Patients with cystic fibrosis who were administered a tracer amount of [14C]spermidine excreted 11--13% of the radiolabel within 72 hr whereas normal controls excreted 60--76% of the radiolabel within 72 hr. Spermine excretion was positively correlated with increased pathology as assessed by the National Institutes of Health (NIH) clinical score, whereas urinary putrescine and spermidine levels were negatively correlated with increased pathology.

Since prolactin, like the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, induces ornithine decarboxylase and plasminogen activator activities, biochemical markers of a trophic response, this hormone might likewise promote neoplasia. To test this theory, rats were initiated with a hepatocarcinogen followed by six weeks of ovine prolactin. This regimen caused hepatomegaly and the development of enzyme-altered foci. Promotion with prolactin for 23 weeks further increased the numbers of enzyme-altered foci. We suggest that prolactin is an endogenous tumor promoter for chemically initiated cells.

In White Leghorn chickens, 0.5 ng/kg of thyrotropin-releasing hormone (TRH) increased ornithine decarboxylase activity in the rostral lobe of the adenohypophysis without any alteration of enzyme activity in the caudal lobe. In rats, administration of TRH (250 ng/100 g body weight) increased thyroid-stimulating hormone (TSH) release but did not increase ornithine decarboxylase activity in the adenohypophysis. However, surgical thyroidectomy performed on rats resulted in declining levels of T3 and T4 in the plasma, an increase in the plasma level of TSH, and a twofold increase in ornithine decarboxylase activity in the adenohypophysis. In the chicken, administration of both methimazole and thyroxine caused elevated ornithine decarboxylase activity in the rostral and caudal lobes of the adenohypophysis. The hormone stimulation of ornithine decarboxylase activity, an early indicator of new biosynthesis, is suggested as a marker to study control mechanisms in the adenohypophysis.

Cellular proliferation and differentiation of the mammalian mammary gland requires a medley of hormones including the anterior pituitary hormone, PRL. Recent evidence extends the role of PRL as a mammalian mitogen to cells in several physiological systems not directly involved in reproductive functions, such as liver and lymphocytes. PRL administration induces biochemical markers expressed during the G 1 phase of cell cycle and activates DNA synthesis in rat liver. Chronic PRL treatment causes hepatomegaly, reflecting its stimulation of the proliferative process. In vitro , a lactogen-dependent cell line, the Nb2 rat node lymphoma cell, serves as a useful paradigm to study PRL action on mitogenesis. These cells, when cultured in the presence of lactogens, proliferate in a dose-dependent manner. The effects of various pharmacological agents on discrete phases of the cell cycle may be readily assessed in these cells since PRL-stimulated entry into cycle is signalled by an elevation of ODC activity at 6 hr and entry into S-phase at 6–12 hr. The parallel effects of phorbol ester tumor promoters and PRL on cell cycle progression in Nb2 lymphoma cells and in hepatic proliferation suggest that PRL may likewise mediate proliferation in aberrant growth conditions such as neoplasia. The data presented support the hypothesis that PRL is capable of promoting hepatocarcinogenesis. Its chronic administration after a hepatic initiating agent stimulated the development of histochemical and biochemical markers characteristic of preneoplasia. Further, the effect of PRL was comparable to that of the hepatocarcinogen when either was administered alone. Thus, hyperprolactinemia may serve to promote the development of hepatic tumors. Phorbol esters are thought to promote tumorigenesis by directly activating PKC. In the Nb2 lymphoma cell model, tumor promoting phorbol esters mimic the effects of PRL. Similarily, PRL-stimulated enzyme induction in liver is mirrored by phorbol ester treatment, and inhibitors of PKC block PRL-stimulated mitogenesis in Nb2 cells. Further, PRL or TPA administration to rats causes translocation of PKC activity from the hepatic cytosol to the membrane fraction, reflecting kinase activation. Therefore, PRL activation of PKC appears to be a physiological phenomenon of general significance, occurring as the result of lactogen receptor stimulation and serving to transmit intracellular signals linked to the regulation of mitogenesis. Further study is required to more fully define the scope of PRL-mediated mitogenic actions as well as its effects on the expression of differentiated products in tissues and cells.

Purified ornithine decarboxylase (EC 4.1.1.17, ODC) transamidated with four putrescine moieties on four glutamine residues through the action of transglutaminase (EC 2.3.2.13, TGase) purified from guinea pig liver, when added to isolated rat liver nuclei, stoichiometrically increased the activity of RNA polymerase I (EC 2.7.7.6). Te increase was relative to the pmoles of purified conjugated ODC added to the reaction and could be reinitiated after the reaction had plateaued by the further addition of ODC-putrescine conjugate. The kinetics of the reaction suggest that the ODC-putrescine conjugate was not reused but degraded after each initiation. Otherwise, the rapid plateau would not be observed. The repeated addition of 278 pmoles of purified ODC-putrescine conjugate to rat liver nuclear preparations containing 200 μg total protein consistently stimulated the incorporation of 600–700 pmoles UMP/mg protein. We suggest that ODC transmidated by its product putrescine may be the posttranslationally modified 65,000 M r protein which has been reported by several laboratories to serve as a labile subunit of RNA polymerase I.

Polyamine biosynthesis and accumulation were studied during the early development of the nudibranch Phestilla sibogae. Ornithine decarboxylase activity increases over 40-fold in the first 4 days of embryogenesis, with the maximum (400 pmole of 14CO2/30 min/mg of protein) occurring between Days 3 and 4, the time of most rapid growth. Putrescine-stimulated S-adenosyl- l -methionine decarboxylase activity is not detectable until the second day of development and attains maximal activity (100 pmole of 14CO2/30 min/mg of protein) at Day 4. The pattern of spermidine-stimulated S-adenosyl- l -methionine decarboxylase activity is similar. Putrescine and spermidine double in concentration between Days 0 and 6. Adults contain fairly high levels of putrescine and spermidine, similar to prokaryotes. The increase in the polyamine biosynthetic enzymes and the accumulation patterns of the polyamines correlate well with data from other early developmental systems and support the hypothesis of key roles for the polyamine pathway in the control of growth processes.

Quaking mutants and jimpy mutants of mice have known deficiencies of myelination of the central nervous system, as well as lesser involvement of the peripheral nervous system. Both mutants also have altered polyamine synthesis and accumulation, particularly in the hindbrain and spinal column. The ratio of spermidine/spermine, which generally is higher in tissues with high rates of biosynthetic activity, was significantly lower in the mutants as compared to their normal siblings. In quaking mutants, 5 months of age, the spermidine concentration of hindbrain and spinal column was 60% that of controls. In contrast, the decreased spermidine/spermine ratio in jimpy mutants resulted from a marked increase in the spermine concentration in both forebrain and hindbrain. Alterations in the spermidine/spermine ratio could lead to reductions in the biosynthetic potential of the brain during development.

Several prototype macrophage (MO) populations were compared for differences in ectoenzyme phenotype and polyamine content. Resident peritoneal MO and Corynebacterium parvum (CP)-activated peritoneal MO expressed unique ectoenzyme phenotypes, while bone marrow derived MO (BMDMO), obtained from stem cells after 7 days in culture with colony stimulating factor, and thioglycollate (TG)-elicited peritoneal MO exhibited a similar ectoenzyme phenotype. All of the MO populations, however, differed in polyamine accumulation patterns. These results suggest that ectoenzyme phenotypes do not serve as completely selective markers of MO differentiation. Moreover, BMDMO do not resemble steady state tissue peritoneal MO but appear to resemble inflammatory MO in several respects. Therefore activated BMDMO do not appear to provide an accurate model system for their continued use in studies to characterize the development of resident tissue MO.

Ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, may be controlled by a cAMP-dependent mechanism. This hypothesis was investigated in the adrenal medulla of the rat. Exposure of rats to cold (4°, 2 hr) leads to increased cholinergic nerve transmission and to a 10- to 20-fold increase in cAMP levels in the medulla within 30 min. The cAMP level returned to normal within 2 hr of the initiation of cold exposure. Ornithine decarboxylase activity was elevated within 1 hr of cold exposure and by 4 hr was increased 10- to 20-fold. We also studied the effects of various drugs which were agonists and antagonists of the cAMP response to cold exposure in the medulla. Aminophylline (200 μmoles/kg), an inhibitor of phosphodiesterase activity, caused a large, rapid increase in the cAMP level followed by an increase in ornithine decarboxylase activity similar to that after cold exposure. Injection of a cholinomimetic drug, carbamylcholine (4.1 μmoles/kg), caused a 10- to 15-fold increase in cAMP within 20 min and a 10-fold elevation in ornithine decarboxylase activity within 2.5 hr. Pretreatment of the rat with the nicotinic receptor antagonist, mecamylamine (15 μmoles/kg), greatly reduced the carbamylcholine-induced rise in both cAMP levels and ornithine decarboxylase activity. Mecamylamine administered alone did not alter either cAMP levels or ornithine decarboxylase activity. Administration of reserpine (16 μmoles/kg) also resulted in an early rise in cAMP concentration in the adrenal medulla and a concomitant increase of ornithine decarboxylase activity. Cyclic AMP has been postulated to exert its effect on cellular metabolism via the activation of a cAMP-dependent protein kinase. Varying doses of reserpine from 1.6 to 16 μmoles/kg yielded a 1:1 relationship between the degree of activation of cAMP-dependent protein kinase(s) and the induction of ornithine decarboxylase. We feel that evidence from this and other laboratories supports the hypothesis that ornithine decarboxylase may be controlled by cAMP-dependent protein kinase(s).

Administration of TCDD produced a significant decrease in the serum concentration of prolactin (PRL) detected in rats after 4 hr compared to pair-fed vehicle controls and noninjected controls. This effect of TCDD was reversed by pimozide, a dopamine receptor antagonist. These data suggest that TCDD decreased the release of PRL from the adenohypophysis either by a direct effect on the gland or by altering the dopamine concentration in the median eminence (ME). Concentrations of TCDD from 5 to 500 ng/ml had no direct effect on the ability of the adenohypophysis to secrete PRL in vitro. However, the dopamine concentration increased to 3.24 +/- 0.07 ng per ME in TCDD-treated rats compared to 2.81 +/- 0.08 ng in vehicle controls. This is a dramatic alteration in the dopamine concentration, since the dopamine is being measured in the portal circulation which exhibits a rapid turnover. The rate constant of dopamine depletion after alpha-methyl-p-tyrosine and the turnover rate were also significantly elevated in the ME of TCDD-treated rats. These data provide the first biochemical evidence for a hypothalamic site of action of TCDD. Since dopamine is inhibitory to PRL release from the adenohypophysis, increased ME steady-state concentrations and turnover of this catecholamine may be responsible for the decreased concentration of serum PRL detected within 4 hr of TCDD injection. Thus, one of the early modes and sites of action of TCDD is to elevate the dopaminergic activity of the tuberoinfundibular nucleus. A hypothalamic site of action for TCDD may result in a number of the endocrinological effects known to be produced by exposure to TCDD.

The pharmacological characteristics of the myocardial adrenoceptor of the mouse have been examined during embryogenesis by measuring ornithine decarboxylase (ODC, EC 4.1.1.17) induction. 2 A four fold elevation of ODC activity was observed after isoprenaline (10 mg/kg, s.c.), and enzyme activity was increased two to three fold following adrenaline (1 mg/kg, s.c.) or terbutaline given by direct injection to the foetus (10 microgram/500 mg). 3 Pretreatment with the beta-adrenoceptor antagonist, propranolol (10 mg/kg), totally blocked the increase in ODC activity. 4 Elevation of myocardial ODC activity was not inhibited by metoprolol, a relatively specific beta-adrenoceptor antagonist, at a dose of 10 mg/kg. 5 Since the increase in ODC activity was blocked by a beta-adrenoceptor antagonist (propranolol) and enzyme activity was stimulated by terbutaline, a beta 2-agonist, we conclude that beta 2-adrenoceptors are selectively coupled to the regulation of murine cardiac ODC activity following catecholamine stimulation.

Transglutaminase (TGase) activity was measured during cell cycle progression in Chinese hamster ovary (CHO) cells synchronized by release of quiescent cultures and in CHO cells synchronized by mitotic shake off. In cells released from quiescent cultures, a greater than 2-fold increase in TGase activity occurred within 3 h of stimulation, followed by a rapid decline and a second and third peak of activity at 6 and 9 h after stimulation. The increase in TGase activity was not inhibited at any time measured by the administration of cycloheximide. Further, there was a significant increase in TGase activity detectable at 1, 5, and 6 h in CHO cells exposed to actinomycin D at time zero. TGase activity in CHO cells was not increased by the addition of either dibutyryl cAMP or 8-bromo-cAMP whereas ornithine decarboxylase (ODC) activity was increased at all times measured by the administration of cAMP analogs. These data suggest that TGase and ODC are regulated by separate hormonal pathways during cell cycle progression. In addition, ODC is induced in CHO cells by a transcriptional mechanism whereas TGase appears to be activated. In order to determine if the biphasic increase in TGase exhibited in G1 of cell cycle was the result of two populations of cells progressing through G1, we assessed TGase activity in CHO cells synchronized by mitotic shake off, which produces a greater than 95% cell synchrony. Again, there were two major peaks of TGase activity at 3 and 5 h, respectively. However, there was a marked difference in the expression of TGase within the first 2 h after release from mitosis. During this period, there was a marked and significant decrease in TGase activity within 1 h to a value about 35% of the time zero value. Another G1-specific marker, ODC, also exhibited a marked decline in activity within the first hour, followed by a single peak of expression between 4 and 5 h in these cells. Both enzymes have high activity during G2 phase of cell cycle as previously reported for ODC. Both enzymes are rapidly decreased after release from mitosis. The peak expression of both TGase and ODC suggest they catalyze important enzymatic events required for cell cycle progression.

The total amounts of type I and type II cytoplasmic cyclic AMP-dependent protein kinase activities were measured in various tissues of intact rats and rats subjected to castration, hypophysectomy, or adrenalectomy. After castration, the total amount of type I activity decreased rapidly in classifically steroid-responsive tissues such as the ventral prostate and levator ani muscle and less rapidly in the liver. After hypophysectomy and adrenalectomy, type I activity in the liver decreased to the same extent as after castration. Type I activity could be maintained in the ventral prostate and levator ani muscle at control levels by the daily injection of dihydrotestosterone. Furthermore, after post-castration regression of the prostate for 3 days, three daily subcutaneous injections of dihydrotestosterone resulted in a complete restoration of type I activity toe the intact level. The amount of type II activity was not altered by any of the experimental ablations. This study provides evidence linking steroid action to the ability of steroid-responsive tissues to maintain a substantial activity of type I cyclic AMP-dependent protein kinase.

Prolactin is an important growth modulatory hormone in fetal and adult tissues. Its administration stimulates enzymatic markers of the G 1 phase of cell cycle in rat liver and other tissues. To determine the effects of prolactin administration on hepatic DNA synthesis (S phase), rats received prolactin at 12 hour intervals for 48 hours and DNA synthesis was assessed by [ 3 h]-thymidine incorporation. Prolactin administration stimulated DNA synthesis 2-4 fold above controls in the livers of adult and weanling animals. Increased incorporation of radiolabel was associated with the nucleus of hepatoparenchymal cells. These data support the hypothesis that prolactin may be a physiological regulator of hepatic DNA synthesis. Further, since stress stimulates prolactin secretion, we suggest that prolactin may participate in the hepatic compensatory hyperplasia elicited by the stress associated with partial hepatectomy.

It appears that the induction of ornithine decarboxylase regulates the rate of ribosomal RNA synthesis as well as regulating the rate of synthesis of polyamines. Further, ornithine decarboxylase, in most cases, is induced after a significant activation of cAMP-dependent protein kinase. We propose a model for the process of hypertrophy based on studies of a considerable number of mammalian growth systems. The mechanism of parallel regulation of polyamines and RNA appears to be initiated by the direct effect of ornithine decarboxylase on RNA polymerase I.

The nuclear content of conjugated polyamines increased during rat liver regeneration. Conjugated polyamines isolated from the acid-precipitable fraction of nuclei required peptide bond hydrolysis for release of the parent compounds. The most striking change occurred in conjugated putrescine which fluctuated in a biphasic manner; maximal nuclear levels 12-fold and 25-fold above those of sham-operated controls were achieved at 4 and 42 hr after hepatectomy, respectively. Conjugated spermidine and spermine increased 3- and 2-fold respectively within 4 hr and remained high throughout the 48 hr studied. When expressed on the basis of mg of nuclear protein, the maximal conjugated putrescine increased 19-fold, conjugated spermidine increased 2-fold, and conjugated spermine decreased by 50%. Therefore, the spermidine and spermine conjugates may be of a more constitutive nature whereas the large changes in the nuclear conjugation of putrescine associated with the onset of growth may play a regulatory role. The nucleus also contained transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13), an enzyme shown in vitro to conjugate polyamines covalently to proteins. The specific activity of the nuclear enzyme increased rapidly after partial hepatectomy to a level 3-fold above control at 4 hr and 7-fold above control at 42 hr. The increased conjugating activity resulted from an increase in detectable maximal velocity and not a change in affinity of the enzyme for putrescine (Km congruent to 0.4 mM). There was also a 3-fold increase at 42 hr in the number of nuclear amine acceptor sites present to which radiolabeled putrescine could be conjugated by endogenous enzyme.

Histologic evaluation of endomyocardial biopsy specimens is the current method of monitoring rejection after cardiac transplantation. Unfortunately, this technique gives a discontinuous evaluation of the recipient immunologic status. A noninvasive marker of immunologic activation and of allograft rejection that would permit a more continuous monitoring than the biopsy technique would be clinically useful. Urinary polyamine excretion reflects cellular proliferation or degeneration and, as a marker of cellular metabolic activity, may also reflect lymphocyte proliferation and organ rejection. From July 1985 to December 1986, urinary polyamines were studied in 18 patients during hospitalization for heart and heart-lung transplantation. Endomyocardial biopsy was performed twice a week and histologic rejection was characterized by standard criteria. Urinary specimens were collected daily and analyzed for polyamines by high-pressure liquid chromatography. Concentrations of acetylputrescine and total urinary polyamines were significantly higher before the 20 rejection episodes than before the 80 biopsies yielding negative results. So that their clinical usefulness could be evaluated, an elevation of polyamines and a daily level variability of 28% or more was chosen to indicate increased metabolic cellular activity and to predict rejection in the next 8 days. On the basis of these definitions, the sensitivity of polyamine assays to predict rejection was 85%, the specificity 88%, and the positive predictive value 79%. Therefore, serial measurements of urinary polyamines may provide daily information on the recipient’s immunologic status after cardiac transplantation.

IGF-I stimulated the mitogenesis of Nb2 cells in the presence of suboptimal mitogenic concentrations of prolactin (PRL, 0.01 or 0.1 ng/ml). In the presence of 1 ng/ml or 10 ng/ml, concentrations of PRL that produced a maximal or near maximal mitogenic response in Nb2 cells, the addition of insulin-like growth factor-I (IGF-I) at 5 or 10 ng/ml did not further enhance mitogenesis. This effect was selective for IGF-I since IGF-II, epidermal growth factor (EGF), insulin, transforming growth factor-β (TGF-β) or platelet-derived growth factor (PDGF) did not stimulate mitogenesis in the presence or absence of PRL. That the ability of IGF-I to stimulate mitogenesis of these cells required PRL was suggested by the ability of PRL antiserum to block the IGF-I effect. Monoclonal antibody to IGF-I reduced the mitogenic response to one identical to PRL alone. In the presence of a suboptimal mitogenic concentration of PRL, IGF-I was an equally effective comitogen when added at 0 time or at 6 h after PRL stimulation, suggestive of an effect of IGF-I in late G 1 phase of the cell cycle. Effects of IGF-I on ornithine decarboxylase, a G 1 enzymatic marker, were compatible with this interpretation. In order to be assured that IGF-I exerted its action through a receptor-mediated process, we evaluated the presence of IGF-I receptors on Nb2 cells. Receptors were present which bound IGF-I>IGF-II⪢ insulin. The IC 50 was 35 nM for IGF-I, 280 nM for IGF-II and 875 nM for insulin. The K d for IGF-I was 5.45 pM and the B max was 1.32 pmol/2 × 10 6 cells. These observations suggest that IGF-I may facilitate DNA synthesis and mitogenesis in PRL-dependent Nb2 cells in the same way that IGF-I and EGF serve as progression factors for BALB/c 3T3 mouse fibroblasts [Leof, Van Wyk, O'Keefe & Pledger (1983), Exp. Cell Res. , 147 , 202–208; Leof, Wharton, Van Wyk & Pledger (1982), Exp. Cell Res. , 141 , 107–115; Campisi & Pardee (1984), Molec. cell. Biol. , 4 , 1807–1814]. That PRL is required as a necessary component for IGF-I mitogenic action is demonstrated by the lack of any effect of IGF-I in the absence of PRL.

Prolactin (PRL) activated protein kinase C (PKC) in a dose dependent manner in rat aortic smooth muscle. Aortic strips incubated with sub-nanomolar concentrations of ovine PRL for 25 min. at 37° C showed a significant stimulation of PKC activity in both cytosolic and particulate fractions. This activation could be blocked using either anti-PRL antibodies or 1-(5- isoquinolinesulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The results further support the role of PKC in the signal transduction pathway for PRL action and suggest that this activation may be involved in vascular smooth muscle function.

Serial samples of plasma and serum were collected in the morning and afternoon from cancer patients, along with 24-h urine specimens. Values for serum and plasma samples taken at the same time from the same patient differed little, suggesting that either procedure is acceptable for polyamine analysis. Increases in concentrations of putrescine and spermidine in serum and plasma correlate well with such increases in 24-h urine samples. Spermidine concentrations in sera were consistently about 10-fold lower than corresponding urine values. Putrescine concentrations were 10to 100-fold different. The variation in putrescine values may be due to its more active metabolism by diamine oxidase, an enzyme known to be present in serum. Concentrations of polyamines in serum and urine increased in response to effective chemotherapy.

The administration of aminophylline results in rapid increases in cyclic AMP in the adrenal medulla, adrenal cortex, liver, and kidney of the rat. The injection of theophylline results in a similar increase in cyclic AMP in the liver of the rat. In all instances, these increases are followed by 4- to 2-fold elevations of ornithine decarboxylase activity. The generality of this phenomena suggests that ornithine decarboxylase activity is regulated by an increase in cyclic AMP.

We have developed a reproducible model of cardiac hypertrophy in conscious, unrestrained dogs after recovery from surgical trauma. The model has many potential applications due to the availability of non-stressful blood sampling from four arterial and/or venous vascular locations. Samples of blood for biochemical or pharmacological measurements were obtained from the carotid and femoral arteries as well as the pulmonary artery and inferior vena cava. Left ventricular hypertrophy up to 128% of the non-operated control animals was produced at 96 h post-intraluminal aortic coarctation. Inflation of a balloon in the descending aorta increased outflow resistance and resulted in hypertrophy. Hemodynamic parameters of cardiac function were obtained via a Swan-Ganz cardiac output catheter located permanently in the pulmonary artery. Complications observed in the dog model were minimal and mortality did not occur during the experimental period. This animal model employing multiple implanted catheters for blood sampling plus the ability to impose aortic coarctation in the unrestrained animal provides a flexible model system for biochemical and pharmacological research.

Prolactin stimulates a hepatotrophic response similar to that caused by phorbol esters or partial hepatectomy in rats. Since phorbol esters, which activate protein kinase C, mimic prolactin action in liver, the relationship between prolactin administration and subsequent hepatic protein kinase C translocation was assessed. Prolactin administration rapidly stimulated a 4-fold elevation of membrane protein kinase C activity. The effect of prolactin on hepatic protein kinase C was specific for lactogenic hormones but could be duplicated by phorbol esters. Further, an increase in serum prolactin was demonstrated subsequent to partial hepatectomy and preceding hepatic protein kinase C translocation. Therefore, translocation of hepatic protein kinase C appears important for hepatic proliferation in response to prolactin administration and to partial hepatectomy.

Increased circulating epinephrine levels paralled cardiac hypertrophy in response to aortic constriction in the dog. Venous levels of epinephrine were significantly elevated at 24, 48, 72 and 96 hours postcoarctation. There were not significant arterial or venous alterations in norepinephrine levels. Dogs administered propranolol daily had no detectable hypertrophy 96 hours postcoarctation. We suggest that an increased epinephrine level may contribute to the physiological trigger(s) for cardiac hypertrophy.

In addition to its growth regulating properties, new evidence reviewed here by Diane Haddock Russell demonstrates that prolactin has important immunoregulatory properties. In immune-compromised dwarf mice, prolactin restores immunocompetence. Human lymphocytes have prolactin receptors and mitogen-stimulated lymphocytes make and secrete a prolactin-like activity. Prolactin can stimulate the activation of nuclear protein kinase C in spleen and liver isolated nuclear preparations. This activation is blocked by prolactin receptor monoclonal antibody, suggesting that there is a receptor-mediated activation process in the nucleus. The discovery of the ability of prolactin and growth factors to activate nuclear protein kinase C may constitute a breakthrough in our understanding of how these hormones regulate trophic responses.