The cardiac sarcoplasmic reticulum-glycogenolytic complex A possible effector site for cyclic

Cardiac sarcoplasmic reticulum-glycogenolytic complex, isolated as a single peak on sucrose density gradient, may function as a “compartmented” effector site for cyclic AMP resulting in modulation of both glycogenolysis and calcium transport. The conversion of phosphorylase b to a is stimulated by ATP and inhibited by protein kinase inhibitor. Cyclic AMP alone stimulated neither phosphorylase b to a conversion nor calcium uptake. An inhibitor of adenylate cyclase depressed both calcium uptake and phosphorylase activation and both functions were subsequently stimulated by micromolar concentrations of cyclic AMP. Endogenous phosphorylation of sarcoplasmic reticulum was also inhibited by adenylate cyclase inhibitor and the inhibition was reversed by cyclic AMP. These results suggest that the sarcoplasmic reticulum of cardiac muscle is an internal effector site for cyclic AMP which may regulate both calcium and metabolism. It appears that cyclic AMP formation in vitro is not the rate-controlling step in the activation sequence.     

Uptake of transferrin-bound zinc by human lymphocytes.

Uptake of transferrin-bound zinc was stimulated in phytohemagglutinin-treated human lymphocytes as compared to untreated lymphocytes. Stimulation of zinc uptake depended upon the concentration of phytohemagglutinin and was maximal for the first hour after addition of phytohemagglutinin to lymphocyte cultures. Thereafter, increased zinc uptake declined until approximately basal levels were reached 5 hr after addition of phytohemagglutinin. Stimulation of zinc uptake was insensitive to sulfhydryl reagents, but was decreased by KCN, actinomycin D, aurin tricarboxylic acid, and by lowering the incubation temperature. Two compounds, NaF and poly-l-ornithine, were found to markedly increase zinc uptake over that seen with only phytohemagglutinin. Additionally, compounds known to increase cellular levels of cyclic AMP, such as epinephrine, histamine, serotonin, glucagon, and prostaglandin E₁, as well as 8-bromo-cyclic AMP and dibutyryl-cyclic AMP, also increased uptake of transferrin-bound zinc by phytohemagglutinin-stimulated lymphocytes.     

Modulation of peripheral blood mononuclear cell cyclic adenosine monophosphate levels by human very low density lipoprotein

Normal human plasma very low density lipoprotein (VLDL) can inhibit mitogen-induced lymphocyte DNA synthesis. Since early events in lymphocyte activation (e.g., cyclic nucleotide metabolism) are thought to influence the magnitude of later events (e.g., [³H]thymidine uptake) we designed the current studies to compare the effects of VLDL on these two cellular processes. Two separate effects of VLDL on peripheral blood mononuclear cell (PBM) cyclic adenosine monophosphate (AMP) metabolism were observed at VLDL concentrations which inhibit phytohemagglutinin (PHA)-induced [³H]thymidine uptake. VLDL suppressed the early, transient increase in PBM cyclic AMP which occurs within minutes of the addition of mitogen. VLDL exposure also stimulated a delayed (greater than 24 hr) and spontaneous increase in PBM cyclic AMP levels which corresponded temporally with progressive cellular refractoriness to mitogen stimulation. If mitogen-induced lymphoproliferation is influenced by early changes in cyclic nucleotide metabolism, as claimed by some investigators, then perhaps the ability of VLDL to modulate intracellular cyclic AMP levels may explain some of the antiproliferative properties of this bioregulatory lipoprotein.     

Inhibition of mitogen-induced lymphocyte transformation by local anesthetics.

Chlorpromazine (CPZ) and lidocaine were added to cultures of mouse spleen cells stimulated by concanvalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). Concentrations of CPZ greater than 5 x 10(-6)M and concentrations of lidocaine greater than 2 x 10(-3)M totally inhibited the mitogenic responses to all four mitogens. Minimal inhibitory concentrations of neither drug interferred with cell viability as determined by trypan blue uptake or 51Cr release. The effects were totally reversed by the removal of the drugs from the culture. Addition of the drug at intervals after mitogen exposure demonstrated that the inhibited event occurred relatively soon after exposure to the mitogen. For example, the addition of lidocaine or CPZ more than 24 hr after Con A stimulation had no effect on tritiated thymidine incorporation. Elevated concentrations of cyclic AMP, cyclic GMP (or their derivatives) or calciunown membrane active actions of these drugs and the rapid reversibility of the effect strongly support the idea that the local anesthetics act on the surface membrane of lymphocytes. Binding of radiolabeled Con A or LPS to lymphocyte membranes in the presence of lidocaine or CPZ was not inhibited. The possibility exists that CPZ and lidocaine disorganized cell membranes so as to interfere with the surface membrane elaboration or action of a second messenger, or interfere with cell-cell interactions.     

Sterilization of Listeria monocytogenes by guinea pig peritoneal exudate cell cultures

Non-glass-adherent cells (lymphocytes) of peritoneal exudates from guinea pigs infected with bacillus of Calmette-Guerin (BCG), stimulated in vitro by specific (tuberculin) or nonspecific phytohemagglutinin P (PHA) mitogens, conferred on glass-adherent cell (macrophage) cultures from BCG-infected, or homologous, noninfected guinea pigs the ability to sterilize Listeria monocytogenes. Lymphocytes from noninfected guinea pigs, stimulated by mitogens, had little or no activity in this test system, although the adherent monolayer cells were seen to be “activated” by morphologic criteria when PHA was employed.Phagocytosis of the bacteria was inhibited in sterilizing macrophage-lymphocyte cultures even after washing of the cultures had eliminated most of the cell clusters seen in activated cultures. However, the monolayers, before and after washing, were found to produce a soluble, filtrable factor(s) which sterilized the listeria. This activity was detectable as early as 17 hr in mixed-cell culture filtrates, and 42-hr monolayers continued to generate this active material after repeated washings with fresh medium up to 72 hr.No listeria-sterilizing activity was found in filtrates of mitogen-stimulated nonadherent lymphocyte cultures without macrophages, and such filtrates were not active in stimulating macrophage monolayers to produce the material although the cells in such monolayers were seen to manifest increased surface adherence and spreading characteristic of “activated” macrophages. Also, such culture filtrates were shown not to antagonize the antibacterial activity of listeria-sterilizing filtrates.Preliminary characterization of the listeria-sterilizing material revealed the following: (a) a molecular weight of 50,000 or more; (b) stable at 56 °C for 30 min in medium containing serum; (c) bound to the bacterial cells at 0 °C; (d) inactivated by the strong reducing agent, dithiothreitol, and partially reactivated by H₂O₂ oxidation; (e) partially antagonized by deoxyribonucleic acid; (f) inactive against two species, of salmonella; (g) not inhibited or enhanced by listeria-agglutinating antibodies.     

Two categories of lymphocyte unresponsiveness to phytohemagglutinin

Peripheral lymphocytes from healthy subjects, sarcoidosis and influenza patients were studied in vitro by measurement of the tritiated thymidine uptake of unstimulated and phytohemagglutinin. (PHA) stimulated cells. When the mitogen induced metabolic response is defined as the ratio between thymidine uptake by stimulated and unstimulated cells (stimulation index), PHA responsiveness was significantly decreased in both diseases and varied inversely with the level of isotope incorporated by unstimulated cells (p = 0.0002). The uptake of isotope by unstimulated cells from influenza patients was significantly increased (p = 0.0001). Isotope incorporation by mitogen stimulated cells from the same patients did not differ significantly from controls (p = 0.0925). In contrast, the impaired PHA responsiveness of lymphocytes from sarcoidosis patients was associated with levels of isotope incorporation in unstimulated cell cultures similar to those observed in healthy controls (p = 0.6444). These observations suggest that two different mechanisms may be responsible for low lymphocyte PHA stimulation indices associated with disease states. Methods are presented for minimizing variation of replicate observations and identification of both categories of lymphocyte unresponsiveness.     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Interaction of Bacillus Calmette--Guérin-activated macrophages and neoplastic cells in vitro II. The relationship of selective binding to cytolysis

5′-Methylthioadenosine (MTA), a degradation product of S-adenosylmethionine, inhibits DNA and protein synthesis as well as cellular proliferation in human lymphocyte cultures stimulated with mitogens, antigens, or allogeneic cells. MTA (10^?3M) inhibited [³H]Tdy uptake in PHA- or Con A-induced transformation greater than 95%, and inhibited the uptake of both [³H]Tdy and [¹⁴C]Leu to the same degree in lymphocytes stimulated with PPD or allogeneic lymphocytes. MTA inhibition was dose dependent, inhibition being lost when exogenous levels reached approximately 10^?5M. The inhibitory effects of MTA were not produced by cytotoxicity since MTA-inhibited cells washed free of this compound could be stimulated at least as well as uninhibited cells. Understanding the mode of action of MTA and the mechanisms controlling its metabolism may lead to new approaches for regulating cellular proliferation.     

Inhibition of lymphycyte reactivity in vitro by autologous polymorphonuclear cells (PMN).

The influence of autologous polymorphonuclear cells (PMN) on lymphocyte reactivity was investigated by monitoring the uptake of tritiated thymidine by unstimulated, phytohemagglutinin (PHA)-stimulated, and fetuin-stimulated lymphocytes in vitro. Addition of PMN at PMN-to-lymphocyte ratios (P:L) of 0.5 to 2.0 progressively inhibited lymphocyte reactivity. Soluble extracts, obtained by sonication and ultracentrifugation (100,000g for 90 min) of PMN, also inhibited lymphocytes. The PMN-derived inhibitor(s) is noncytotoxic, heat labile (56 °C for 60 min), resistant to freeze-thawing (20 cycles), and appears to be nondialyzable. Inhibition was more marked when the factor was added at the initiation of lymphocyte cultures than when added with the tritiated thymidine 24 hr prior to cell harvest. Thus thymidine released by PMN which diluted the radiolabeled nucleotide and degradation of the tritiated thymidine did not explain these results. Lymphocytes incubated for 3 days in the medium containing the inhibitor reacted normally to PHA following washing, indicating that inhibition was reversible. These results suggest that a PMN-derived lymphocyte inhibitor(s) may modulate lymphocyte-mediated immune reactivity.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110^Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34^cdc2 synthesis, and cyclin A synthesis. In control cultures, p110^Rb phosphorylation was detected within 24 hr of PHA stimulation; p34^cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c-fos, IL-2, and IL-4 mRNAs expression, and IL-2 receptor (p55) expression, were only marginally affected, if at all, in PHA-stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL-2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL-2/IL-2 receptor interaction but prior to p110^Rb phosphorylation and other key regulatory events signaling G₁/S transition. © 1993 Wiley-Liss, Inc.     

Interaction between prostaglandins and cyclic amp in the gastric mucosa

Possible mechanisms accounting for the inhibition of acid secretion by prostaglandins were studied using cells dispersed from canine fundic mucosa by enzymes and enriched in the content of parietal cells by elutriation. The accumulation of ¹⁴C-aminopyrine (AP) was used as an index of parietal cell response to stimulation. PGE₂ inhibited histamine-stimulated AP uptake, with 50% inhibition (ID₅₀) found at 10 nM, but did not block the response to carbachol, gastrin, or dibuturyl cyclic AMP. PGE₂ did, however, inhibit aminopyrine uptake stimulated by carbachol and gastrin when the response to these agents was potentiated by histamine. PGE₂, at namomolar concentrations, also inhibited histamine-stimulated cyclic AMP production. When mucosal cells were treated with only PGE₂ at concentrations above 1 μM, stimulation of cyclic AMP production was found. In cell separation studies with the elutriator rotor, PGE₂ appeared to stimulate cyclic AMP production primarily in nonparietal cells.Prostacyclin (PGI₂) and two stable analogues, 6_β-PGI₁ and the 16-phenoxy analogue (5_α)5,9-epoxy-16-phenoxy-PGF₁, also specifically inhibited histamine-stimulated AP accumulation. PGI₂ required relatively high concentrations for this effect (ID₅₀ = 1 μM), whereas the 16 phenoxy derivative was much more potent in its inhibition of histamine-stimualted AP accumulation (ID₅₀ = 10 nM), with this difference probably accounted for by the rapid degradation of PGI₂ compared to the stable 16-phenoxy analogue. All three of these prostanoids also inhibited histamine-stimulated cyclic AMP production. As was found with PGE₂, at high concentrations and in the absence of histamine PGI₂ and PGI₁ also stimulated cyclic AMP production. However, the 16-phenoxy analogue failed to stimulate cyclic AMP production either in the parietal cell enriched fractions or in the nonparietal cell fractions.These data indicate that PGE₂ and prostacyclin analogues are potent, direct and specific inhibitors of histamine-stimulated parietal cell function and that it is the inhibition, rather than the stimulation, of cyclic AMP formation that is linked to the antisecretory actions of the prostanoid compounds.     

Regulation of immune responses. I. Effects of cyclic AMP and cyclic GMP on immune induction.

Agents that raise intracellular cAMP levels (dibutyryl cyclic AMP, aminophylline, adenosine and butyric acid) increase the magnitude of an in vitro primary humoral immune response when added at 10^?3M during the first 12 hr of a 108 hr culture. Under the same conditions, cGMP has no direct effect but inhibits cAMP-mediated stimulation. DbcAMP (10^?3M or 10^?4M), present from 0 to 12 hr, also increases the number of cytotoxic lymphocytes in CBA/J (H-2^k) spleen cell cultures stimulated in a one-way mixed lymphocyte reaction with DBA/2J (H-2^d) spleen cells. The dbcAMP effect is antigen-dependent in both humoral and cell-mediated immunity and antigen-specific in the case of humoral responses.     

The cardiac sarcoplasmic reticulum-glycogenolytic complex. A possible effector site for cyclic AMP.

Cardiac sarcoplasmic reticulum-glycogenolytic complex, isolated as a single peak on sucrose density gradient, may function as a "compartmented" effector site for cyclic AMP resulting in modulation of both glycogenolysis and calcium transport. The conversion of phosphorylase b to a is stimulated by ATP and inhibited by protein kinase inhibitor. Cyclic AMP alone stimulated neither phosphorylase b to a conversion nor calcium uptake. An inhibitor of adenylate cyclase depressed both calcium uptake and phosphorylase activation and both functions were subsequently stimulated by micromolar concentrations of cyclic AMP. Endogenous phosphorylation of sarcoplasmic reticulum was also inhibited by adenylate cyclase inhibitor and the inhibition was reversed by cyclic AMP. These results suggest that the sarcoplasmic reticulum of cardiac muscle is an internal effector site for cyclic AMP which may regulate both calcium and metabolism. It appears that cyclic AMP formation in vitro is not the rate-controlling step in the activation sequence.     

Characteristics of calcium accumulation by lymphocytes and alterations in the process induced by phytohemagglutinin

Calcium uptake by normal human lymphocytes was found to be a saturable process which was competitively inhibited by manganese indicating the existence of a carrier-mediated mechanism for calcium uptake. Exchange diffusion was not observed, Phytohemagglutinin (PHA) significantly stimulated calcium uptake within minutes after treatment. The increased uptake was attributed to a decreased K_m for the proposed membrane carrier rather than to an increased V_max. Also PHA did not stimulate a normally unused exchange diffusion process, nor did it affect calcium efflux. Uptake by both unstimulated and PHA-treated lymphocytes was not influenced by magnesium or by cycloheximide or actinomycin D.     

The elicitation of phytoalexins by Ca2+ and cyclic AMP in carrot cells

Addition of calcium ionophore A23187 or dibutyryl cyclic AMP (dBcAMP) to carrot (Daucus carota L.) cell culture induced the production of 6-methoxymellein, a phytoalexin of carrot, in a dose-dependent manner. Several reagents known to suppress the cytoplasmic calcium concentration appreciably inhibited elicitor-promoted phytoalexin production in carrot cells. The addition of elicitor to the carrot culture caused a rapid increase in the intracellular level of cyclic AMP. Treatments of the cells with theophylline or cholera toxin stimulated the biosynthesis of 6-methoxymellein even in the absence of elicitor. These observations suggested that Ca²⁺ and cyclic AMP participate as second messengers in the regulation of 6-methoxymellein production in cultured carrot cells. Addition of verapamil to carrot cell culture markedly inhibited 6-methoxymellein production when it was added within 30 min after elicitor-treatment of the cells, but no inhibitory effect was observed after 60 min. The results suggest that these messengers function in an early stage of the elicitation process. Carrot cells which were previously treated with verapamil accumulated only small amounts of 6-methoxymellein following the addition of dBcAMP. In contrast, cells incubated initially with dBcAMP accumulated the phytoalexin at levels comparable to the control when verapamil was added to the culture.     

Prostaglandins and cyclic AMP stimulate creatine kinase synthesis but not fusion in cultured embryonic chick muscle cells

Cyclic AMP levels have been measured in cultures derived from 12-day-old chick embryonic muscle. A rise in concentration was found after the onset of myoblast fusion. Cells cultured at a medium Ca²⁺ concentration of 0.1 μM did not fuse and exhibited only a small rise in cyclic AMP concentration during culture. Addition of 1.4 mM Ca²⁺ to these cells after 50 h in culture caused rapid, synchronous fusion with a concomitant rise in cyclic AMP levels. Indomethacin, an inhibitor of prostaglandin synthesis, did not inhibit fusion, but inhibited the rise in cyclic AMP concentration. Indomethacin-treated cultures exhibited lower creatine kinase levels, though no change in the ratio of the three isoenzymes was observed. Addition of prostaglandins E₁ and E₂ to indomethacin-treated cultures overcame this inhibition. We propose that prostaglandin synthesis is a consequence of the stimulation of myoblast fusion and that via cyclic AMP it stimulates protein synthesis.     

Prostacyclin analogues inhibit canine parietal cell activity and cyclic AMP formation

To assess further the mechanism by which prostacyclin inhibits acid secretion, the actions of two stable prostacyclin analogues on parietal cell function and cyclic AMP formation were tested using enzymatically dispersed cells from canine fundic mucosa. Accumulation of ¹⁴C-aminopyrine (AP) was used as an index of parietal cell response to stimulation. The 16-phenoxy derivative of PGI₂ inhibited accumulation of AP stimulated by histamine (10 μM), with 50% inhibition (ID₅₀) at 10 nM. 6_β-PGI₁ also inhibited the action of histamine (ID₅₀ 0.5μM) but failed to block stimulation by carbachol or the dibutyryl derivative of cyclic AMP (dbcAMP). In similiar concentrations to those producing inhibition of histamine-stimulated AP accumulation, the 16-phenoxy analogue and 6_β-PGI₁ inhibited histamine-stimulated cyclic AMP generation by parietal cells. At 100 fold higher concentrations, 6_β-PGI₁ stimulated cyclic AMP formation, presumably in non-parietal cells. Even in high concentrations the 16-phenoxy analogue failed to increase cyclic AMP formation by mucosal cells. These data indicate that the stable prostacyclin analogues are potent, direct inhibitors of histamine-stimulated parietal cell function and that it is the inhibition, rather than the stimulation, of cyclic AMP formation that is linked to the antisecretory actions of these prostanoid compounds.     

Calcium-mediated effects of calcitonin on cyclic AMP formation and lymphoblast proliferation in thymocyte populations exposed to prostaglandin E 1

The calcitonin (SCT) from salmon ultimobranchial bodies which (like mammalian calcitonins) lowers the plasma calcium concentration in mammals can also affect cyclic AMP (cyclic adenosine 3′,5′-monophosphate) metabolism and proliferation of lymphoblasts in normal and prostaglandin E₁ (PGE₁)-treated rat thymocyte populations in three different ways. In the first case, low concentrations (0.5–5.0 ng per milliliter) of SCT lower (by a calcium-mediated process) the ability of PGE₁ to transiently increase cyclic AMP synthesis, but the reduced surge of cyclic AMP production is still ample to stimulate lymphoblasts in the cell population to initiate deoxyribonucleic acid (DNA) synthesis. Secondly, these low SCT concentrations affect the eventual progression of the PGE₁-stimulated, DNA-synthesizing lymphoblasts into mitosis by a calcium-mediated process. Depending on the extracellular calcium concentration and the magnitude of the initial increment in the intracellular cyclic AMP content, SCT can either promote or inhibit the progression of the stimulated cells into mitosis. SCT's third action is a rapid (within 5 minutes), calcium-independent elevation of the cellular cyclic AMP content in otherwise untreated thymic lymphocyte populations exposed to a very high concentration (100 ng per milliliter) of the hormone. This early, transient rise in the cyclic AMP level is followed by a calcium-dependent increase in lymphoblast proliferation. An attempt is made to interrelate and explain the different actions of SCT on cyclic AMP metabolism and mitogenesis.