PHYTOCHEMICAL ANALYSIS, VOL. 3, 14-19 (1992)

A Simple Assay for Adenosine 3’,5’-Cyclic Monophosphate (Cyclic AMP) and its Application to the Study of Cyclic AMP Production in the Lichen Himantormia lugubris

Carlos Vicente” and Jose L. Mateos Department of Plant Physiology, Faculty of Biology, Complutense University, 28040 Madrid, Spain

This paper describes an accurate, specific and sensitive high performance liquid chromatographic method for the identification and quantification of adenosine 3’,5’-cyclic monophosphate (cyclic AMP). Recovery of the cyclic nucleotide has been estimated to be about 82.6% after the extraction procedure. By using this method, the cyclic nucleotide has been identified in the lichen Himantormia lugubris. Production of cyclic AMP is achieved in the dark by this lichen species and is inhibited by white light. This inhibition is reversed by adding 3-(3’- dichloropheny1)-1,l-dimethylurea or adenosine triphosphate to the incubation medium. The effects of red light and far-red light on cyclic AMP accumulation by the lichen have been investigated.

Keywords: Himantormia lugubris; adenylate cyclase; cyclic AMP; high performance liquid chromatography; phosphodiesterase; phytochrome.

INTRODUCTION

Many physiological functions of adenosine 3‘,5‘-cyclic monophosphate (cyclic AMP) have been reported in plants. Cyclic AMP reverses catabolite repression of several enzymes in eukaryote plant cells, even that produced by urea (Vicente and Legaz, 1985); this is in contrast to that found for urea-sensitive operons in prokaryotes (Sanzey and Ullmann, 1976). Cyclic AMP induces swelling of etiolated oat protoplasts (Chung et al . , 1988) and facilitates diffusion of some organic anions across the plasma membrane (Avalos and Vicente, 1987). The latter is also effected by red light and, in this way, both cyclic AMP and dibutyryl cyclic AMP replace the light requirement for anion permea- tion. A similar replacement has been described for betacyanin synthesis in Amaranthus paniculatus (Rast et al. , 1973). These effects seem to be related to the phytochrome function in plant cells. In addition, the nucleotide produces elicitation of phytoalexins in carrot cells (Kurosaki et al . , 1987) and activates protein kinases other than protein kinase C, which is specifi- cally activated by diacylglycerol (Cockroft, 1987).

The availability of accurate and simple methods to identify and quantify cyclic AMP in plants is clearly of importance. Radioimmunoassay (RIA) of 2‘Oacetyl- cyclic AMP derivatives is the most usual method employed to estimate cyclic AMP in both plant and animal extracts (Frandsen and Krishna, 1976; Harper and Brooker, 1975). High performance liquid chroma- tographic (HPLC) procedures have been described which include sample pretreatment and separation but, since such methods are complicated, considerable losses of metabolites during processing may occur (Van Ockelen et at., 1982).

* Author to whom correspondence should be addressed

0958-0344/92/010014-06 $05 .00 0 1992 by John Wiley & Sons, Ltd.

This paper describes a simple procedure for the analysis of cyclic AMP by reversed phase HPLC and its application to the resolution of specific physiological questions, such as the effect of light on the level of this nucleotide in a lichen species, Himantormia lugubris.

EXPERIMENTAL ~ ~~

Plant material and incubation conditions. H. lugubris (Hue) Lamb was collected from the soil on King George Island (Antarctica). Samples were air-dried and stored in polythene bags in the dark at 5°C until required. Samples of 0.5 g of air- dried thalli were floated on 12 mL 0.1 M sodium acetate- acetic acid buffer (pH 6.8) for 3 h at 26°C in the dark. Where indicated, 10 m M ethylenediamine tetraacetic acid (EDTA), 10 mM glucose, 0.5 mM cyclic AMP, 2 mM adenosine triphos- phate (ATP), 10 pM 3-(3’-dichIorophenyl)-l,l-dimethylurea (DCMU) or 30 pM filipin were added to the buffer. Where appropriate lichen samples on acetate buffer, or acetate buffer containing either DCMU or ATP, were incubated for 3 h at 26°C in white light (18W Philips Daylight TLD 54 fluorescent tubes; photon flux rate 70 prnollm’ls). Alternatively lichen samples on acetate buffer, or acetate buffer containing filipin or EDTA, were floated for 3 h at 26°C in the dark but given 5 min pulses of red light (R) or 5 min pulses of R followed by 5 min pulses of far-red light (FR) at the start of each hour of incubation. R (75 pmol/m’/s) was provided by 18 W Sylvania Gro-lux fluorescent tubes, the output from which was filtered through a 3.0mm layer of Plexiglass 52 (Rohm). FR (75 pmol/m2/s) from 150 W Osram incandescent lamps was filtered through one layer of red Plexiglass 52 and a 3.0mm layer of blue Plexiglass 64 (Rohm). Determinations of photon flux rates were made at plant level in all cases. The spectral distribution of relative energy was measured using a Spectra-Scan spectro- radiometer and the results are shown in Fig. 1.

Received 18 March I991 Accepied (revised) 20 August I991

HPLC ANALYSIS OF CYCLIC AMP 15

R n

d 'I- al g 0.2s J U .- = I I \ I I 5 1 , ,),k - , I al L I )

500 550 600 650 700 750 800

h ( n m ) Figure 1. Relative spectral energy distribution of red (R) and far-red (FR) sources.

Preparation of plant extracts for HPLC analysis. After incu- bation, thallus samples were washed with distilled water, gently dried with filter paper and macerated in a mortar with 25 mL chloroform for 15 rnin at room temperature to remove lichen phenolics (Vicente et al., 1983). Homogenates were filtered through Whatman No. 3 filter paper and solid resi- dues were dried in an air flow. The dry residues were macerated again with 4.0 mL distilled water and centrifuged at 27,000 x g for 30 min at 2°C. Where indicated, 32.16 ng cyclic AMP was added to the residues prior to the water extraction. Supernatants were stored and precipitates were extracted again as above. Mixtures of both supernatants were precipitated with an equal volume of acetonitrile. Precipitates were removed by centrifugation at 27,000 X g for 30 rnin at 2°C and the supernatants dried in an air flow. Solid residues were resuspended in 2.0 mL methanol, filtered through Millipore GS filters (0.22 pm) and injected onto the chroma- tographic column. Alternatively, mixed supernatants were incubated at 37°C for 30min with one unit of activator- insensitive phosphodiesterase 3',5'-cyclic nucleotide (Sigma Chemical Co., St. Louis, MO) prior to the addition of acetonitrile; subsequently the extraction procedure was per- formed as above.

Chromatographic analysis. Samples and standards were chro- matographed using a Varian 5000 liquid chromatograph equipped with a Vista CDS 401 computer. Chromatographic conditions were as follows: reversed phase column (300 mm x 4 mm i.d.) packed with MicroPak MCH-10; load- ing - 10 pL sample; mobile phase - methanol: acetic acid: water (80: 0.5 : 19.5 v/v) isocratic elution; pressure - 22 atm; temperature - 20°C; How rate ~ 0.2 mL/min (as deduced f rom Van Deemter equation); UV detector - h = 254 nm; absor- bance units at full scale - 0.005.

Standards. Adenosine, cyclic AMP, cyclic GMP, cyclic UMP, AMP and ATP (Sigma Chemical Co.) were used as standards. Chloroform (2mL) was added to each standard (2.0mg) and the suspensions were dried in an air flow, resuspended in 2.0 mL distilled water, to which 2.0 mL aceto- nitrile was added, and dried again. Each dried residue was redissolved in 5.0 mL ethanol, filtered through Millipore GS

filters and injected onto the column. Where indicated, 0.2 mL standard solutions were added to the samples obtained from plant material.

RIA of cyclic AMP. Alternatively, cyclic AMP was measured by RIA. For this purpose, lichen samples of about 0.5 g were homogenized with 5.0 mL trichloroacetic acid (TCA) (6%) at 4"C, and centrifuged at 2,500 x g for 15 min. Supernatants were extracted four times with water-saturated diethyl ether and the aqueous phase dried at 80°C. The solid residues were redissolved in 0.5 M acetate buffer (pH 6.2) and cyclic AMP was derivatized to 2'-O-acetyl-cyclic AMP with triethylamine and acetic anhydride (Harper and Brooker, 1975). Cyclic AMP was estimated by RIA (Frandsen and Krishna, 1976) by using RIANENTM CAMP '*'I-RIA kits from Dupont. Non-specificity (measured against cyclic GMP) has been esti- mated as lower than 0.01% (Avalos and Vicente, 1987).

RESULTS

HPLC separation of cyclic AMP and its analogues

Figure 2(A) shows the Van Deemter equation obtained by using the various standards. A flow rate of 0.2 mL/ min was the optimum for the separation of cyclic nucleotides with the maximum plate number, and the chromatographic separation of the standard cyclic nucleotides is shown in Fig. 2(B) at this flow rate. Quantification of cyclic AMP was linear in the range of concentrations 0-0.2 pg of mass injected (Fig. 2C). A peak with retention time identical to that of standard cyclic AMP was obtained from H. lugubris thalli rehyd- rated in acetate buffer for 4 min in the dark (Fig. 3A). This peak alone increased when lichen samples were loaded with standard cyclic AMP (Fig. 3B), whereas peaks well distinguished from that of this nucleotide appeared when samples were loaded with either cyclic GMP or cyclic UMP (Figs. 3C and 3D).

Yield of the extraction procedure and accuracy and specificity of the method

Aqueous homogenates of lichen thalli, after chloro- form extraction, were supplied with 32.16 ng cyclic AMP (chromatographically estimated) and the final extracts were analysed by HPLC. The overall recovery of cyclic AMP was estimated at 82.6% (Table 1). A part of the supplied nucleotide seemed to be degraded to adenosine (11.4%) and about 6% of the total sup-

Table 1. Recovery of exogenously supplied cyclic AMP to aqueous extract of H. lugubris thalli, as estimated by HPLC, and comparison of both chromatographic and RIA methods

Cyclic AMP Analysis Addition to (ng/g dry Recovery method aqueous extract thalli)' ( % I HPLC none 1.28 ? 0.16 - HPLC 32.16 ng 27.84 f 0.32 82.59 RIA none 1.49 ? 0.18 -

"Values are the mean of three different experiments assayed in duplicate f standard error.

16 C. VICENTE AND J. L. MATEOS

I cGMP

A

I I I I I 0 0.2 olr 0.6 0.6 in

ATP flow rate U ( m l / m i n 1 (19.10 1

cUMP (12.20) 1 A U D

cGMP ( 1 0 . 5 0 1 b (14.A)

adenosine ( 25.10 1

8

d - 1 I I I I 1

0 10 20 30 40 5 0 t ime (min 1 ''Or 6 00 1 600 -

600-

200 - /* C

I I I 0.05 010 015 0.20

0. /* I

0 mass injected ( n g )

C

I I I 0.05 010 015 0.20

0. 0

mass injected ( n g )

Figure 2. (A) Van Deemter equation for the nucleotides used as standards in the HPLC procedure; (6) chromatographic trace for the separation of standards by HPLC. (C) direct calibration of cyclic AMP by HPLC: y=3378.15x- 1.60; r2=0.99.

plied was lost foliowing acetonitrile precipitation and subsequent centrifugation, possibly bound to protein precipitate. Comparison of the chromatographic method with the radioimmunoassay resulted in differ- ences smaller than 15% (Table 1). The identity of the cyclic AMP peak (retention time, 14.4min) was con- firmed by incubating aqueous extracts of lichen samples with phosphodiesterase for 30min at 37°C. The reac- tion was stopped by adding acetonitrile to the mixtures and methanolic extracts were chromatographed. Only the peak previously identified as cyclic AMP com- pletely disappeared and a peak with a retention time of 25.10 min, tentatively identified as adenosine, quan- titatively increased, as shown in Fig. 4.

Effect of light on cyclic AMP accumulation in H . lugubris thalli

Figure 5 shows that cyclic AMP accumulated in H. lugubris thalli floated on acetate buffer in the dark, although a decrease of the nucleotide concentration

L

2 I \

1 1 - 0 5 10 15 20 25

0 5 10 15 20 25 -30 t i m e ( m i n 1

0 5 1 0 1 5 2 0 2 5 1 I I I I I j

0 5 10 15 20 25 30 time (min 1

Figure 3. Identification of cyclic AMP in cell-free extracts from H. lugubris rehydrated by floating on acetate buffer for 4 min in the dark (A) or loaded with cyclic AMP (61, cyclic GMP (C) or cyclic UMP (D) (peaks labelled 1 =cyclic GMP, 2=cyclic AMP, 3 = adenosine and 4 = cyclic UMP).

was observed after the third hour of thalli incubation under white light. The amount of cyclic AMP in the thallus fell to a minimum after the first hour of culture. The effect of light was completely reversed by including 1 0 p ~ DCMU in the acetate-containing media. The amount of cyclic AMP strongly increased from the first hour of culture and then slightly decreased, although concentrations of cyclic AMP at any particular time were always higher than those found in the dark (Fig. 5) . The effect of light was also reversed by adding 2.0 mM ATP to the acetate-containing medium after 1 h of light treatment in the absence of the nucleotide. Amounts of cyclic AMP were always lower than those found when DCMU was used.

3 A B

2 n

I 1 1 -

0 5 10 15 20 25 1 1 1 1 1 1

0 5 10 15 20 25 30 t i m e ( m i d

Figure 4. Effect of incubation of aqueous extracts, obtained from thalli incubated on acetated buffer for 4 min in the dark, with phosphodiesterase for 30 min at 37°C. where (A) is the control without enzyme treatment and (B) after enzyme incuba- tion (peaks labelled 1 =cyclic GMP, 2=cyclic AMP and 3 =adenosine).

HPLC ANALYSIS OF CYCLIC AMP 17

A

w .c rn .- i 1 >r

.u L 1

\" rn C Y

0. SI U 0

0

0

1-

d

0 ? 2 3 h o u r s

Figure 5. Time course of cyclic AMP accumulation by H. lugu- bris thalli floated on acetate buffer in the dark ( 0 ) or in white light (0). The acetate medium was supplemented with 10 VM DCMU (A ) or 2 mM ATP ( 0 ) where indicated. Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols.

- 2 . 2 m g / g a ,

Phytochrome mediation of cyclic AMP accumulation

In order to investigate whether the quality of light promoted changes in cyclic AMP concentration thallus samples were floated on acetate buffer in the dark, but 5 min pulses of R, or 5 rnin R and 5 rnin FR pulses were given at the start of each hour of incubation. As is shown in Fig. 6, red light strongly increased production of cyclic AMP for the first hour of thalli incubation, although it decreased thereafter. When FR was given after R pulses, values of cyclic AMP concentration were close to those found in thallus samples incubated in the dark. The action of red light was also negated by adding 30 pM filipin to the acetate-containing media.

The unexpected decrease of cyclic AMP concentra- tion after the first R pulse could be due to an enhanced phosphodiesterase activity. In order to test this hypoth-

I I I

1 2 3

h o u r s Figure 7. Time course of cyclic AMP accumulation by H. lugu- bris thalli floated on acetate buffer containing 0.5 mM cyclic AMP (B), or on 10 mM glucose containing the nucleotide (0) . The disappearance of cyclic AMP from acetate buffer was measured ( A ) as well as the time course of cyclic AMP accumulation by using thalli floated on acetate buffer containing 10 mM EDTA (0). Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols.

esis, two different experiments were performed. When thallus samples were floated in the dark on an acetate- containing medium to which 0.5 mM cyclic AMP had been added, the cyclic nucleotide rapidly disappeared from the medium during the first hour of thallus incuba- tion, but the levels in the thalli strongly increased

0 1 2 3 h o u r s

Figure 6. Effect of 5 rnin R pulses ( B f or 5 rnin R + 5 min FR pulses ( A ) at the beginning of each hour of incubation of H. lugubris thalli floated on acetate buffer in the dark. When 30 VM filipin was added to the acetate medium (0). only red treatment was performed. Values are the mean of three repli- cates. Vertical bars give standard error where larger than the symbols.

I I I

1 2 3 h o u r s

Figure 8. Time course of cyclic AMP accumulation by H. lugu- bris thalli floated on acetate buffer containing 10 mM EDTA. Lichen thalli received 5 min R pulses ( B ) or 5 min R + 5 min FR ( A pulses at the start of each hour of incubation. Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols.

18 C. VICENTE AND J. L. MATEOS

(Fig. 7). However, cyclic AMP slowly disappeared from the medium and strongly decreased in the thallus upon further periods of incubation. This latter decrease was eliminated by adding 10 mM EDTA to acetate/cyclic AMP-containing media. Uptake of cyclic AMP by lichen thalli was markedly decreased by including 1 0 m ~ glucose in the media. In a second experiment, thallus samples were floated on an EDTA-acetate medium and treated, with R and R-FR pulses (as described previously) during incubation. In this case, R treatment continuously increased the amount of cyclic AMP, whereas levels obtained after R-FR treatment were almost identical to those found in the thalli follow- ing incubation in the dark (Fig. 8).

DISCUSSION ~

The HPLC procedure described for the separation of cyclic AMP from other cyclic nucleotides and AMP provides sufficient accuracy and resolution even when applied to lichen-cell-free extracts (Figs. 2 and 3). Losses during extraction procedure can be reduced to about 17%. This loss includes both cyclic AMP degraded by endogenous phosphodiesterase (liberated during cell-free extract preparation) and that which presumably binds to cellular structures and proteins and is thus removed by centrifugation. The chromato- graphic method estimates 15% less cyclic AMP than that of RIA but, in contrast, it is more rapid, safer and cheaper. The peak with a retention time of 14.4 min has been fully identified as cyclic AMP since this peak almost completely disappears after incubation of aqueous lichen extracts with commercial phosphodies- terase. Recovery of enzymatically degraded cyclic AMP as adenosine is explained on the basis of the phosphatase activity contained by the commercial enzyme preparation.

When this method was used to analyse extracts from H . lugubris thalli incubated under different light con- ditions, it was shown that continuous darkness main- tained a slow but positive production rate of cyclic AMP. This production is clearly from the ATP formed during the respiration process. Contrary to expec- tations, white light, which supports active algal photo- synthesis, completely nullifies cyclic AMP production, although such synthesis is greatly increased by adding DCMU to the incubation medium (Fig. 5 ) . Lichens are C3 plants (Hill, 1976) and it may be supposed that ATP production coupled to non-cyclic electron transport is used by kinases from the reductive pentose phosphate cycle. On this basis, adenylate cyclase is dependent upon an excess of ATP which is not used for hexose phosphate formation. Thus, the size of the ATP pool is the limiting factor in adenylate cyclase activity (Can- tore et al., 1980). When DCMU acts as an uncoupling factor of non-cyclic electron transport (Avron, 1980), the reductive pentose phosphate cycle fails since glyceraldehyde-3-phosphate dehydrogenase has insuf- ficient NADPH to function. Thus, ATP production coupled to cyclic electron transport could be used preferentially by an adenylate cyclase to produce cyclic AMP. Metabolic regulation of this enzyme, performed by intermediate metabolites of the Calvin cycle on adenylate qclase, seems not to occur since H . lugubris thalli, floated on acetate buffer in while light, are able

to restore adenylate cyclase activity after an exogenous supply of ATP. The enhancing effect of white light on cyclic AMP concentration has also been reported by Brown and Newton (1981) and Brown et al. (1989) for Phaseolus seedlings. However, this action of light was directly related to a loss of substrate affinity by phos- phodiesterase rather than to a general photosynthetic effect.

When the light regime consisted of R pulses for 5 min, phytochrome action was revealed by the enhancement of production of cyclic AMP during one hour of darkness after the first R pulse. This effect was reversed by FR (Fig. 6). However, phytochrome action is not absolutely required for the synthesis of cyclic AMP, since its production is performed in the dark as well as in R-FR treatment (Figs. 5 and 6). Thus, the enhancing effect of R can be explained as an activation of adenylate cyclase rather than a de novo synthesis of this enzyme, in a similar way to that described for Evernia prunastri (Avalos and Vicente, 1987). Phytochrome must be involved in this action, as demonstrated by the inhibitory action of filipin on the increasing effect of R on cyclic AMP concentration (Fig. 6). Filipin is a polyene antibiotic that interacts with membrane steroids to which phytochrome possibly binds (Avalos and Vicente, 1989; Roth-Bejerano and Kendricks, 1979). However, the R effect is not main- tained for time periods longer than 1 h; after this time, second and third pulses of R strongly decrease the amount of cyclic AMP in the thallus. In order to explain this transient effect, Avalos and Vicente (1987) suggested that Pf, (far-red absorbing form of phytoch- rome) sequentially activates both adenylate cyclase and phosphodiesterase. This was confirmed by using fors- kolin, an activator of adenylate cyclase (Seamon and Daly, 198l), and theophylline, an inhibitor of phospho- diesterases (Amrheim and Filner, 1973) in E. prunastri (Vicente and Avalos, 1989). Thus, the decrease in cyclic AMP concentration after 1 h R treatment could be due to an enhanced phosphodiesterase action (Fig. 6). The effect of a supply of EDTA to H . Zugubris thalli from media containing exogenous cyclic AMP supports this hypothesis. Exogenous nucleotide rapidly per- meates into lichen cells, reaching the maximum concen- tration value at 1 h treatment, but after this time the amount of cyclic AMP in thalli rapidly decreases (Fig. 7). This last effect is impeded by EDTA, since this compound behaves as an inhibitor of Mg2+-dependent phosphodiesterase by chelating the inorganic cation (Rutter et aZ., 1973). In contrast, Mg2'-dependent ade- nylate cyclase can be protected against the chelating action of EDTA by sequestering Mg2+ in a stable ATP-Mg-enzyme complex (Londos and Preston, 1977). Similar results are obtained when the exogenous supply of cyclic AMP is substituted by R pulses: in this case the loss of intracellular cyclic AMP after 1 h of thalli incubation is avoided (Fig. 8). The transient increase in the content of cyclic AMP after R pulses effecting phytochrome has also been found by Janistyn and Drumm (1975) for mustard seedlings although cotyle- dons were preferentially affected whereas the increase in the amount of nucleotide measured for the axis was slow but continuous for the complete time of experi- mentation (about 2 h).

It is interesting to note that glucose nullifies cyclic AMP uptake by H . lugubris thalli (Fig. 7). This fact can

HPLC ANALYSIS OF CYCLIC AMP 19

be explained as a reciprocal inhibition between glucose and cyclic AMP of their respective sites of permeation in the plasmalemma (Legaz and Vicente9 1991)2 in a similar way to that found by Peterkofsky (1977) for Escherichia coli.

Acknowledgements

This work was supported by a grant from Direccion General de Investigacion Cientifica y Tecnologica (Spain) PB87 0081. We are indebted to Prof. M. E. Legaz for her constructive discussions.

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