Cyclic AMP,cyclic GMP,and bean rust uredospore germlings

Ten millimolar cyclic AMP (cAMP) or cyclic GMP (cGMP) induced bean rust uredospore germlings to undergo one round of mitosis and to form septa, processes normally associated with appressorium formation. To assess the possibility of cyclic nucleotide regulation of bean rust development, we used an 8-azido-[³²P]cAMP photoaffinity probe to identify three cyclic nucleotide binding peptides. The peptides bound either cAMP or cGMP. The phosphorylation of one peptide in uredospore germling extracts by [γ-³²P]ATP was stimulated by either 1 μM cAMP or cGMP, but only in the presence of 10 mM Na₂MoO₄, a phosphatase inhibitor. Uredospores contain about 1500 and 23 pmol cAMP and cGMP/g dry wt, respectively, as determined by radiobinding assays.     

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.     

Differential effects of dibutyryl cyclic AMP on the growth and morphology of an established human lymphocyte line

Summary RPMI 1788 lymphocytes growtn in semi-suspension culture proliferate as separate cells and in clumps. The addition of 10⁻³ m dibutyryl cyclic AMP (Bu₂cAMP) to the culture resulted in the attachment of the cells to the substratum and a subsequent conversion of a portion of the adherent cells to a fibroblast-like morphology. Growth of the adherent cells proceeded at nearly the same rate as that of the control cells. When the cells cultured in the presence of Bu₂cAMP were periodically disturbed, they remained in suspension and under this condition a distinct inhibition of growth by Bu₂cAMP was observed. Cortisol at 10⁻⁵ m, a concentration having no effect on the proliferation of RPMI 1788 cells, when added to cells cultured in the presence of Bu₂cAMP, prevented cell attachment, caused detachment of already adherent cells and thereby brought about the Bu₂cAMP-mediated inhibition of growth in suspension. At a higher concentration (10⁻⁴ m), cortisol alone reduced the growth rate of RPMI 1788 lymphocytes. Under the combined effects of 10⁻⁴ m cortisol and 10⁻³ m Bu₂cAMP, the proliferation and viability of cells in suspension were significantly lower than in the presence of either agent alone.     

Cyclic GMP-activated channels of the chick pineal gland: Effects of divalent cations,pH, and cyclic AMP

Chick pineal cells maintained in dissociated cell culture express an intrinsic photosensitive circadian oscillator, but the mechanisms of phototransduction in avian pinealocytes are not fully understood. In this study, we have used inside-out patches to examine the characteristics of cyclic GMP-activated channels of chick pinealocytes in more detail, concentrating on the effects of factors known to modulate the secretion of melatonin and/or the function of circadian pacemakers. In most patches, the predominant conductance state was 19 pS in symmetrical 145 mM NaCl. But in some patches, a second cyclic GMP-activated channel with a unitary conductance of 29 pS was also present. The current flowing through cyclic GMP-activated channels was not affected by application of salines containing 1 M Ca²⁺ to the cytoplasmic face of the patch membrane. By contrast, application of 1 mM Ca²⁺ caused a partial reduction in cyclic GMP-activated current at all membrane potentials. Application of 1–5 mM Mg²⁺ ions caused a virtually complete blockade of current at positive membrane potentials, but caused only a small decrease in current at negative membrane potentials. No obvious differences in the gating of cyclic GMP-activated channels were observed in pH 8.2, 7.4 or 6.2 salines. Application of salines containing 100 M, 500 M, or 1 mM cyclic AMP did not cause activation of the channels, but 5 mM cyclic AMP evoked a low level of channel activity. Application of 5 mM but not 100 M cyclic AMP decreased the probability of channel activation caused by 20–100 M cyclic GMP and also increased the percentage of openings to an 11 pS subconductance state. Thus, cyclic AMP acts as a weak partial agonist. Nevertheless, the gating of these channels does not seem to be controlled directly by physiologically relevant changes in intracellular Ca²⁺, pH, or cyclic AMP.     

Similar and opposite effects of dibutyryl cyclic AMP and insulin on glucose metabolism in adipose tissue

The effects of N⁶-2′-O-dibutyryl cyclic AMP on glucose metabolism and lipolysis in fragments of rat epididymal adipose tissue were studied. Measurements were made of glucose uptake, conversion of glucose carbon to CO₂ and tissue fatty acids and glyceride-glycerol, lactate production, and glycerol release. Low concentrations of dibutyryl cyclic AMP (0.1–0.5 mM) increased all parameters of glucose metabolism and inhibited glycerol release in tissue from both normally fed and fasted rats. Higher concentrations of dibutyryl cyclic AMP (3–5 mM) diminished glucose utilization and greatly accelerated lipolysis. Insulin, 50 μunits/ml, accelerated glucose metabolism in the presence of either low or high concentrations of dibutyryl cyclic AMP though the effect of insulin was greatly reduced by 3 mM dibutyryl cyclic AMP. Tissue exposed to concentrations of dibutyryl cyclic AMP which inhibited glucose metabolism (5 mM), then rinsed and reincubated without dibutyryl cyclic AMP, displayed increased glucose utilization. The results of these experiments emphasize the need for caution in interpretation of the effects of dibutyryl cyclic AMP on adipose tissue metabolism and the need for further research to elucidate the role of cyclic AMP in the regulation of glucose metabolism.     

Cyclic nucleotides modulate lipoxygenase activity and reproduction in oomycetes

Previous studies in our laboratory demonstrated that epoxy alcohols produced from C₂₀ fatty acids by lipoxygenase activity are associated with maintaining vegetative growth. We have also shown the specific down-regulation of lipoxygenase activity in reproductively competent oomycetes in response to cues which trigger reproduction. Other workers have suggested that cyclic nucleotides may also play a role in the switch between vegetative and reproductive growth of fungi. Reproductive activity was assayed in Achlya americana. Oogonium production was eliminated or reduced in the presence of cAMP, dibutyryl cAMP (0.5 mM), or the phosphodiesterase inhibitors caffeine (0.1, 1 mM) and theophylline (1 mM) and increased by 0.5 mM cGMP. Levels of cAMP were significantly higher in vegetative mycelium and were increased fourfold by exposure to 0.1 mM caffeine. These data suggest that an accumulation of cAMP inhibits reproductive activity while cGMP promotes it. Lipoxygenase activity was determined in the presence of cAMP, cAMP phosphodiesterase inhibitors, and cGMP to determine the interaction between cyclic nucleotides and lipoxygenase activity. Lipoxygenase activity in reproductive mycelium of A. americana or Saprolegnia ferax was reduced compared to vegetative mycelium. Lipoxygenase activity of cultures grown and starved in the presence of cAMP (0.5 mM) or caffeine (0.1 mM) showed significant increases over the comparably treated reproductive controls. Exposure to cGMP had no affect on lipoxygenase activity in A. americana. These data suggest that cAMP may maintain vegetative growth by maintaining relatively high lipoxygenase activity levels while the reproduction promoting effect of cGMP is not via lipoxygenase activity down-regulation. Adenylate cyclase activity was significantly higher in vegetative, compared to reproductive, mycelium of both A. americana and S. ferax. Elevated adenylate cyclase activity in vegetative mycelium supports the hypothesis that cAMP maintains vegetative growth by maintaining high lipoxygenase activity.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Eucalyptus phylacis</Emphasis> L. Johnson and K. Hill., A critically endangered relict from Western Australia

Summary In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m⁻²s⁻¹) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A₄ isomer (GA₄). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA₄ for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.     

The possible mediation by cyclic AMP of parathyroid hormone-induced stimulation of mitotic activity and deoxyribonucleic acid synthesis in rat thymic lymphocytes

Purified parathyroid hormone (PTH) strongly stimulates the initiation of deoxyribonucleic acid (DNA) synthesis and thereby raises the flow of cells into mitosis in rat thymic lymphocyte populations maintained in vitro. These actions of PTH are potentiated by caffeine and inhibited by imidazole which indicates that the hormonal action is mediated by cyclic adenosine 3′,5′,-monophosphate (cyclic AMP). The feasibility of cyclic AMP being the mediator of PTH action is established by the observation that a low concentration (10^?7 M) of dibutyryl cyclic AMP precisely mimics the stimulatory action of the hormone on DNA synthesis and cell proliferation.     

An Improved, Automated Adenylate Cyclase Assay Utilizing Preparative HPLC: Effects of Phosphodiesterase Inhibitors

Abstract: Analysis of adenylate cyclase (ACase) activity in broken cell preparations usually involves conversion of [α-³²P]ATP to [³²P]cyclic AMP (cAMP) followed by purification of cAMP by liquid chromatographic methods. An automated, preparative reverse-phase HPLC procedure was developed that purifies cAMP rapidly and decreases variability and background. It permits the separation procedure to be validated rapidly prior to use with actual samples, and is readily adaptable for assaying guanylate cyclase, phosphodiesterases (PDE), or a variety of other related nucleotide-metabolizing enzymes. For ACase assays, 4.5% ZnSO₄-10% Ba(OH)₂ is added to the incubation mixture, and following centrifugation, the supernatant is injected on an HPLC apparatus fitted with a Waters Z-Module containing a 10-μ C₁₈ reverse-phase cartridge. Using a mobile phase of 0.15 M sodium acetate-20% methanol (pH 5.0) at a flow rate of 4 ml/min, cAMP is eluted at k′ > 1.25, whereas k′ < 0.5 for all other adenine nucleotides, permitting collection of the cAMP fraction after running the other nucleotides to waste. The method was validated by characterizing dopamine-sensitive ACase in homogenates of striatum from Sprague-Dawley rats. Basal activity (177 ± 16 pmol/mg protein/min), the stimulation by dopamine (186 ± 19 pmol/mg/min), the apparent K_m for dopamine (5.0 ± 1.5 μM), and expected effects of varying magnesium, EGTA, and GTP were similar to available data. However, it was found that isobutylmethylxanthine (IBMX) or theophylline, usually included in the incubation mixture as PDE inhibitors, markedly inhibited the synthesis of cAMP in both the presence and absence of dopamine. A consequence of this inhibition was a marked change in the apparent K_m of dopamine calculated from a Lineweaver-Burk plot. The use of IBMX to inhibit PDEs was compared with an alternate strategy, the addition of excess exogenous cAMP. Simultaneous analysis of PDE and ACase activity was accomplished by including [³H]cAMP in the incubation and quantifying the amounts of [³H]cAMP hydrolyzed and [³²P]cAMP synthesized. Without IBMX, a concentration of 1 mM exogenous cAMP was sufficient to prevent significant loss of [³H]cAMP. In the absence of exogenous cAMP, 0.5 mM IBMX did not completely prevent the breakdown of [³H]cAMP, whereas 2.5 mM IBMX did. Although there was 25% less [³H]cAMP recovered in the presence of 0.5 mM IBMX than with 2.5 mM IBMX, there was no difference in the amount of [³²P]cAMP formed (either with or without dopamine). Moreover, in the presence of IBMX, there was a 20–30% lower synthesis of [³²P]cAMP compared with incubations in which only 1 mM cAMP was used to prevent breakdown of [³²P]cAMP. These data suggest that alkylxanthines, possibly through effects on adenosine receptors, may cause unexpected effects on estimations of dopamine-stimulated ACase. The use of exogenous cAMP as an alternate substrate for PDEs may be one way to obviate these problems.     

Effect of dibutyryl cyclic adenosine monophosphate on active amino acid transport in Streptomyces hydrogenans

The active uptake of 2-aminoisobutyric acid (AIB) and several other amino acids in resting cells of Streptomyces hydrogenans was found to be stimulated by exogenously added adenosine cyclic monophosphate (cAMP). The uptake of glycerol, sorbose, and pyrimidine nucleosides remained unaffected. Among the various cAMP derivatives tested, the dibutyryl derivative was found to be most effective, followed by monobutyryl cAMP, and cAMP. Dibutyryl cGMP was also found to stimulate AIB transport, and its effectivity was as good as that of dibutyryl cAMP. The effect of dibutyryl cAMP is time dependent and attains its maximum after 40–60 min of incubation at 30°C in K-Na-phosphate buffer. Dibutyryl cAMP-dependent transport stimulation has a high temperature coefficient and is prevented by rifamycin SV or chloramphenicol. The rate of leucine incorporation into protein was rapidly increased upon addition of dibutyryl cAMP. Kinetic studies reveal that the stimulation of AIB transport is characterized by an increase in maximum uptake rate and an unaltered apparent Michaelis constant. Analysis of the unidirectional fluxes show that both influx and efflux are enhanced by dibutyryl cAMP. It is concluded that exogenous dibutyryl cAMP stimulates de novo synthesis of certain protein including the transport catalysts for various amino acids.     

Separation of mitogen-induced suppressor and helper cell activities during inhibition of interferon production by cyclic AMP.

The effect of exogenous cyclic AMP on mitogen-induced suppression and enhancement of the in vitro plaque-forming cell (PFC) response and on mitogen induction of immune interferon (also called type II) in cultures was examined. Mitogen induction of immune interferon was quantitatively associated with mitogen-induced suppressor activity, and cyclic AMP blocked both the suppressor activity and the production of immune interferon in mouse (C57B1/6) spleen cell cultures. The evidence is as follows: (a) The concentrations of dibutyryl cyclic AMP that blocked T-cell mitogen (staphylococcal enterotoxin A) suppressor activity were the same as those that blocked mitogen induction of immune interferon. (b) The blocking action of dibutyryl cAMP on both the suppressor and interferon effects of mitogen was a function of the time of dibutyryl cAMP addition to cultures relative to mitogen addition. (c) A dramatic immunoenhancing effect of mitogen occurred in the presence of dibutyryl cAMP under conditions that blocked production of immune interferon. Specifically, mitogen-induced helper cell function is dramatically enhanced in the presence of dibutyryl cyclic AMP, if the mitogen is added to cultures 24 to 48 hr after SRBC and dibutyryl cyclic AMP. Dibutyryl cyclic GMP did not affect the mitogen- or cyclic AMP-induced effects under the conditions of our test system. Under the conditions described here, then, cyclic AMP appears to selectively block suppressor cell activity while allowing or aiding mitogen-induced helper cell activity. It is possible that the immune response is a reflection of the ratio of helper to suppressor activities in the system.     

Increased collagen synthesis in Kirsten sarcoma virus-transformed BALB 3T3 cells grown in the presence of dibutyryl cyclic AMP

Growth of Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells in the presence of dibutyryl cyclic AMP (dbcAMP) resulted in alteration of morphology, inhibition of growth, and increased collagen synthesis as measured by incorporation of ¹⁴C-proline into collagenase-digestible protein. There was an increase in incorporation of ¹⁴C-proline into collagen when expressed not only as dpm per μg DNA or protein, but also as the relative rate of collagen synthesis compared to total cellular protein synthesis, which suggests that an alteration in amino acid transport cannot totally account for the increased incorporation into collagen. The three properties studied were all affected over a concentration range of 0.10 to 1.0 mM dbcAMP, but each had a slightly different dose-response curve. At 0.5 mM dbcGMP or sodium butyrate, there was no affect on growth, morphology, or the relative rate of collagen synthesis indicating specificity for the dibutyryl analog of cAMP. Growth of the parent line, BALB 3T3, was inhibited by 0.5 mM dbcAMP, but the relative rate of collagen synthesis did not increase. These results suggest that although growth, morphology, and collagen synthesis are altered in transformed cells so that they more closely resemble those of the parent line, each property may be regulated independently.     

Changes in enzyme patterns produced by high potassium concentration and dibutyryl cyclic AMP in organ cultures of sympathetic ganglia

—Preliminary experiments had shown that acetylcholine, the putative mediator of trans-synaptic induction of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in vivo, did not lead to an increase in these enzyme activities in mouse superior cervical ganglia kept in organ culture. It was the aim of the present study to evaluate whether increases in tyrosine hydroxylase and dopamine β-hydroxylase evoked by other stimuli such as potassium or dibutyryl cyclic AMP in such an in vitro system are representative for in vivo trans-synaptic induction where changes in the levels of enzymes involved in norepinephrine synthesis or degradation are strictly confined to TH and DBH. In the presence of elevated concentrations of potassium or 5 mm dibutyryl cyclic AMP under organ culture conditions TH and DBH as well as DOPA decarboxylase and monoamine oxidase were significantly (P < 0.025) increased. The increase in total activities of TH and DBH were completely, those of DOPA decarboxylase and monoamine oxidase partially, inhibited by cycloheximide. In the presence of high concentrations of potassium, the total protein content of the ganglia was 28 per cent higher than in culture controls while dibutyryl cyclic AMP had no significant effect. Cycloheximide alone caused the protein content to fall to 70 per cent of that in control cultures. The loss of protein in the presence of cycloheximide was not accompanied by a simultaneous loss of TH, DOPA decarboxylase or monoamine oxidase, but DBH was decreased. Potassium was shown to increase the incorporation of [³H]leucine into TCA-insoluble protein during an early culture period but dibutyryl cyclic AMP showed no such effect. An increase in the rate of incorporation of [³H]leucine into protein was seen in both the control and elevated potassium cultures after 48 h. This increase did not occur in the presence of dbcAMP. The difference in enzyme patterns under conditions of elevated potassium and dibutyryl cyclic AMP and the fact that no changes in the levels of endogenous cyclic AMP were observed during exposure to 54 mm -potassium for a time period sufficient to initiate changes ultimately leading to elevated TH levels argues against the mediation of the potassium-induced enzyme increases by cAMP. Since changes in enzyme patterns caused by potassium and dbcAMP were not similar to patterns seen in vivo under conditions of trans-synaptic induction we conclude that use of this system as an in vitro model for in vivo trans-synaptic induction necessitates great caution.     

The role of cyclic AMP in parathyroid hormone action in the toad bladder

Summary Parathyroid hormone (PTH) inhibited active transport of inorganic phosphate and stimulated an increase in cyclic AMP concentration in the urinary bladder of the toad,Bufo marinus. Active transport of phosphate in the toad bladder was also inhibited by an analog of cyclic AMP (dibutyryl cyclic AMP) and by other drugs (pitressin and theophylline) which increase toad bladder intracellular cyclic AMP concentration. These data support the concept that cyclic AMP may be the mediator of PTH-induced phosphate transport inhibition in the toad bladder.     

Human dermal microvascular endothelial cells in vitro: Effect of cyclic AMP on cellular morphology and proliferation rate

Macrovascular endothelial cells isolated from the human umbilical vein and microvessel endothelium from the newborn foreskin dermis differ in their requirements for optimal growth in vitor. In the presence of 5 x 10^?4 M dibutyryl cyclic AMP (Bt₂cAMP), human dermal microvessel endothelial cell proliferation rate increased to give a cell number of 203% of control values by day 10 in culture. The cells retained their characteristic endothelial cell morphology, reached confluence, and could be serially passaged. Cells grown in the absence of Bt₂cAMP did not proliferate readily and grew in a disorganized pattern. The effect of Bt₂cAMP on microvascular endothelial cell proliferation rate and morphology could be duplicated by cholera toxin (CT) used together with isobutyl methyl-xanthine (IMX). These agents were found to elevate intracellular levels of cyclic AMP in microvascular endothelium over 40-fold. Human umbilical vein cells in culture failed to respond to either Bt₂cAMP or CT together with IMX. The growth-promoting effect of dibutyryl cyclic AMP (Bt₂cAMP) on human foreskin dermal microvascular endothelium in vitro is in marked contrast to the lack of response of human umbilical vein cells. These results provide further evidence of differences in the mechanisms that regulate macro and microvessel endothelial cell proliferation in vitro.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Effects of insulin on diaphragm muscle independent of the variation of tissue levels of cyclic AMP and cyclic GMP.

The hypothesis that effects of insulin are mediated by an increase in cyclic GMP was examined in “intact” and “cut” hemidiaphragm preparations. After preincubation for 30 min, the diaphragms were exposed to insulin (10 mU/ml) for periods of time varying from 2 to 10 min. Then the tissue content of cyclic GMP was measured by radioimmunoassay. Tissue cyclic GMP levels were not altered by the addition of insulin, although the incorporation of d-[U-¹⁴C]glucose into glycogen was strongly stimulated under the same conditions. When cyclic GMP and dibutyryl cyclic GMP in concentrations covering a wide range were added to the medium, the insulin-like effect on glycogen synthesis could not be reproduced. On the other hand, the high concentration of dibutyryl cyclic AMP in the medium failed to suppress the insulin effect on transmembrane transport of l-arabinose under conditions in which the entrance of this nucleotide into a cell was confirmed by a significant reduction in glycogenesis. Our results suggest that the effects of insulin on striated muscle may be unrelated to either cyclic GMP or cyclic AMP.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation

Summary Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O₂-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O₂-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10^–5 M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced. The synthetic analog of atrial natriuretic peptide, atriopeptin III (10^–7 M), greatly elevated cGMP immunoreactivity in the type I cells. On the other hand, sodium nitroprusside (1 mM) elevated cGMP staining mostly in vascular elements of the carotid body in vitro. The data implicate the involvement of cyclic nucleotides in transduction of natural chemosensory stimuli by the type I cells in rat carotid body.     

Preparative solvent partition chromatography of butyrylated cyclic AMP analogues

A simple and effective separation of cyclic adenosine-3′,5′-monophosphate (cAMP) and its butyryl-substituted analogues using partition chromatography on columns of Sephadex gel in isopropanol/0.5 m ammonium acetate (4:1) is described. The technique is suitable for preparative separations as demonstrated by revised uv spectral data obtained on butyrylated cAMP's purified by this technique. In addition, it has analytical utility in that it allows complete separation of N⁶-monobutyryl cAMP from O^2′-monobutyryl cAMP, thereby permitting simultaneous and independent assessment of the rate of acyl substituent hydrolysis from the disubstituted derivative (N⁶,O^2′-dibutyryl cAMP), and this is demonstrated under several conditions.