Pergamon 0031~9422(94)EOO17-M Phymchmistry. Vol. 36. No. 2, pp. 259-262.1994 Copyright 0 1994 Elscvm Sacna Lid

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GASEOUS NITRIC OXIDE STIMULATES GUANOSINE-3’,5’-CYCLIC MONOPHOSPHATE (cGMP) FORMATION IN SPRUCE NEEDLES

SILVIA PFEIFFER, BORIS JANISTYN, GEORG JESSNER, HEIDI PICHORNER and ROBERT EBERMANN+

Institute of Chemistry, University of Agriculture, Gregor MendelstraBe 33, A-l 180 Vienna, Austria

(Receiced in recked form 6 December 1993)

Key Word Index-Picea; Pinaceae; spruce; nitric oxide (NO); cGMP.

Abstract-The influence of externally applied gaseous nitric oxide (NO) on spruce needles in relation to cGMP formation has been examined. Exposure of spruce needles (Picea abies) to gaseous nitric oxide leads to a strong and rapid increase of the cGMP concentration. Depending on the base level of cGMP in the untreated needles, the concentration of cGMP in the NO exposed needles increased up to four degrees of magnitude. The content of adenosine-3’,5’-cyclic monophosphate (CAMP) remained below the detection limit of the HPLC-method used (lo-’ mol I- I). Therefore, it was not subject to further investigation.

Following the recognition of CAMP as an important inter- and intracellular regulator, and as a mediator of many hormonal activities in a number of tissues, at- tention inevitably turned to the role of other cyclic nucleotides including cGMP. cGMP was first identified by Ashman et al. in 1963 [l], and since that time it has been shown to be widely distributed, occurring in most animal tissues. Now the participation of cGMP in the regulation of biochemical processes is as well established as that of CAMP. The role of cGMP as an intracellular mediator depends on its interaction with three intracellu- lar receptor proteins: cGMP-dependent protein kinase, cGMP-regulated ion channels and cGMP-regulated cyc- lic nucleotide phosphodiesterase [2].

cGMP seems to regulate a number of intracellular processes, e.g. vascular smooth muscle relaxation and neutrophil activation by these receptor proteins. A fur- ther role for cGMP has been found in the photo- transduction system of the retina [3-61. Also, the parti- cipation of cGMP in blood vessel relaxation caused by drugs, e.g. nitroglycerin and nitroprusside, and by agents that require the endothelium for their relaxing effects is discussed [7]. In contrast, the occurrence and function of cyclic nucleotides isolated from plants is still a matter of controversy [&lo]. On the other hand, the cGMP content of maize seedlings was determined by GC-MS and found to be in the range of 35-72 pmolg-’ fresh weight [l I].

Two recent discoveries connect nitric oxide (NO) with physiological cGMP-formation: the NO synthase which has recently been found in animal tissue [12], and the

*Author to whom correspondence should be addressed.

stimulating effect of NO on the guanylyl cyclase in different cell tissues [13-161. Therefore NO, a noxious, paramagnetic radical gas, seems to be an important physiological transmitter substance in the metabolism of vertebrates. There have been different target sites for NO already established in animals. Relaxing the smooth muscle, it acts as an ‘endothehum derived relaxing fac- tor-EDRF’. Its role in inflammation, thrombosis, immunity and neurotransmission is also discussed [17-201. NO has a high affinity for the interaction with ferrous haemoproteins, e.g. guanylyl cyclase and haemo- globin. Stimulating the guanylyl cyclase activity in differ- ent cell tissues, releasing thereby cGMP, it functions as an intra- and intercellular second messenger [21, 223. Since NO is also produced in all combustion processes, it is a component of polluted air. Therefore, externally applied NO might have similar functions in plant tissues. Therefore, it seems reasonable to study the influence of NO on spruce needles in relation to cGMP-formation as a model system.

RESUL'l?SANDDlSC.U5SION

Spruce needles were exposed for 10 min to 60 ppm NO in vacuum. This is ca 1000 x larger than NO concentra- tions found on average in polluted air. The samples of spruce needles used for the NO exposure and the control samples have been identical (same tree, sample of needles taken from a randomly mixed collective). Five different trees were included in the investigation. All trees (Picea abies) were grown under the same ecological conditions, but have been of different age. With the exception of one tree, in which no base level of cGMP has been detected, the base level in the other four trees remained the same, i.e. lo-* mol cGMPg- ’ spruce needles (fresh weight).

259

260 S. PFEIFFFR et cd.

samples. The HPLC peak increased to the extent corres- ponding to the amount of internal standard added. Therefore, the identi~~ation of the cCMP peak was established, since the retention time of the internal stand- ard was identical also. In all of the samples no CAMP content could be found, as the level of this cyclic nucleot- ide remained below the detection limit of the HPLC system used.

For further identification ofthe HPLC peak of cGMP, the diluted extracts were incu~ted with phosphodiester- ase I and II for different time intervals. To optimize the reaction condition for phosphodiesterase II (3’-exonuc- lease, EC 3. I. 16.1. from bovine spleen), the needle extracts were diluted 1: 100 and the pH of f was elevated to pH 6.8

Figure 1 shows the increase in the cGMP formation after exposure to NO for 10 min. Under these conditions an enormous increase of cGMP could be observed (ca four magnitude). In the NO untreated sample no cGMP level has been found. Therefore, NO seems to have very similar effects in plants and animals, in which it functions as an activator of the guanylyi cyclase. In comparison with the sample in which already a base level ofcGMP of IO - * mol g - 1 fresh weight has been detected, the increase of cGMP in the NO exposed needles is only up to two magnitudes. For the identification of the cyclic nucleot- ide, an internal standard of cGMP (10e5 mol I-‘), 1: 10 diluted was added to the 1: 100 diluted extracts of the

0 5 10 15 20 2s 30 3s 40

time (min) 0 5 10 15 20 2.5

tme (min)

0 s 10 15 20 2s 30 35 40

time (mb)

-r , * . . .

0 s 10 15 20 25

time fmin)

I I , , ,

0 s 10 15 20 2s

time (min)

Fig. I. HPLC diagrams of the analysed needle extracts contain-

ing cGMP: (A) 1: 100 diluted sample of the control sample; (B)

I : 100 diluted needle extract of sample treated with 60 ppm NO

for 10 min. the cGMP peak (indicated by the arrow) is equivalent toaconcentration of 10-O molg”’ spruce needles(fresh weight).

Fig. 2. (A) Untreated. I : I(K) diluted sample, pH 6.8; (B) 1: 100 diluted sample, treated with 2 units phosphodiesterase II ml -‘.

pH 6.8. at 37 ’ for IO hr: (C) t : 100 diluted sampie, treated with 2

units phosphodiesterase II ml- ‘, pH 6.8, at 37’ for 30 hr (arrows

indtcate the disappearing cGMP peak).

cGMP formation in spruce needles

Table 1. Quantitative comparison of the results obtained by the two independent analytical methods applied: HPLC and RIA

261

cCMP concentration @mol g- ’ spruce needles, fresh weight) in the extracts quantitatively determined by

HPLC RIA

Sample NO-exposed Control NO-exposed Control

1 1.9+0.13 -- 1.2&0.12 - 2 2.5 * 2.00 0.015 f 0.002 2.OkO.21 0.0090 + 0.003 3 2.lkO.16 0.030 f 0.004 1.1+0.11 0.0025 * 0.002 4 3.0 f 0.24 0.022 f 0.006 2.4kO.30 0.0170 f 0.006 5 1.7+0.18 0.010~0.004 1.3kO.17 0.0160*0.004

The data points are means of three measurements.

with 50 mM potassium hydroxide. Two units of phos- phodiesterase II were added to 1 ml of the diluted sample and incubated at 37” for different time intervals (Fig. 2).

The same procedure was applied for incubation of the extracts with phosphodiesterase I (S-exonuclease, EC 3.1.4.1., from Crotalus atrox), which yielded the same results (not shown). After 10 hr the peak became smaller and after an incubation time of 30 hr the peak completely disappeared as a consequence of phosphodiesterase ac- tion. As the extracts contain a lot of phenolic substances, e.g. flavanoids, which have been described as phospho- diesterase inhibitors [23, 241, the reaction conditions for the enzymic treatment are not at an optimum, which results in the slow decrease of cGMP. Radioimmuno- assay has been used as another independent method for cGMP identification. Using a cGMP [lz51] assay sys- tem, the amount ofcGMP in the extracts found by HPLC analysis could be verified (Table 1).

The results show that by external application of gas- eous NO, cGMP formation of spruce needles rapidly increases, which parallels the metabolic effect of NO in animal tissue in certain respects. The described produc- tion method of NO yielded a gas, which should contain only tiny amounts of NOz. This leads to the conclusion that NO and not NO, is responsible for the described cGMP increase. Therefore, NO seems to play a very important role in plants as second messenger. The amount of increase of the cyclic nucleotide as a con- sequence of NO exposure depends on the base level of cGMP found in the investigated needle extracts. With the exception of one tree, in which no ground level was detectable, the base level of cGMP in the needle extracts of the remaining four trees was lo-* mol g- ’ fresh weight. Following NO exposure, the content of cGMP increased to a maximum level which achieved the same value in all of the samples, i.e. lo- 6 mol g- ’ fresh weight.

Therefore, it seems that there might exist a similar NO- dependent regulation system in plants responsible for cellular cGMP levels as has been described for verteb- rates. A significant parallel to vertebrates is also the result that external applied NO only affects the cGMP level, whereas the cyclic AMP concentration in tissue seems to

be unaffected [253. However, so far no NO synthase has been described in plants. On the other hand NO polluted air may also cause elevated ground levels of cGMP in plants.

EXPERIMENTAL

Chemicals. CAMP (adenosine-3’,5’-cyclic monophos- phate), cGMP (guanosine-3’J’cyclic monophosphate), phosphodiesterase I (5’-exonuclease, EC 3.1.4.1., from Crotalus atrox), phosphodiesterase II (3’-exonuclease, EC 3.1.16. l., from bovine spleen) were obtained from Sigma. All other chemicals were of analytical grade (Merck).

Preparation of the extracts from spruce needles for HPLC analysis. Spruce needles (200 g) [Picea abies, L.] were randomly mixed and divided into 2 equal samples. One part was taken for control and the other 1 was exposed to NO in a 5-l desiccator, which had been evacuated before. NO was evolved by addition of an excess of 7.5% H,SO, to 10 mM NaNO, and an excess of (NH,),Fe(SOJ for 10 min. When the reaction was finished, ca 60 ppm of NO was present in the desic- cator. Afterwards 100 ml boiling 0.1 M HCI, contain- ing 1 g ascorbic acid was poured over both samples, and they were homogenized with an UltraTurrax (20000 rpm min- ‘) for 5 min. The homogenates were then allowed to stand for 5 min at loo” in order to hydrolyse all nucleotides except the cyclic nucleotides. After centrifugation at 31000 g for 20 min, the super- natant was decanted, the sediments resuspended with 100 ml of H,O and centrifuged again (3 1000 y, 20 min). Then the fluids were extracted with CHCI, in a separating funnel and allowed to stand overnight. Both extracts were coned to 10 ml under red. pres. and treated with poly- vinylpolypyrrolidone (PVPP) in order to absorb the phenolic components of the extract. An aliquot of the extract was assayed for cGMP-content with an ion- pairing HPLC system [26].

Incubation with phosphodiesterase. For further identi- fication the analysed samples were treated with phospho- diesterase II (EC 3.1.16.1. from bovine spleen) and phos- phodiesterase I (EC 3.1.4.1. from Crutalus atrox). TWO

262 S. PFEIFFER et al.

units of phosphodiesterase II were incubated with the 1: 100 diluted extract for 10 and 30 hr, at pH 6.8 (elev- ation of the pH of the extracts from pH 1 to 6.8 with 50 mM KOH). The same procedure was applied for incubation with phosphodiesterase I (pH elevated with 50 mM KOH to pH 8.8). The enzyme incubated samples were re-analysed by HPLC.

HPLC sysrem. A LKB-liquid chromatograph equipped with a variable UV-spectrophotometric detector and a HP 3396 Integrator was used for ion-pairing HPLC analysis. The sepn was performed on a Nucleosil 120 5C18 column (250x4). The solvent system (isocratic mode) was a mixt. of 4% MeCN in a 0.1 M KPi buffer, pH 7.2, containing 20 mM KBr and 5 mM tetrabutylam- moniumhydrogensulphate (TBAHS). UV absorbance was monitored at 253 nm.

Radioimmunoussa~~. A cyclic GMP [’ 251] assay system (Amersham) was used. The extracts were diluted, so that the amount of labelled cGMP in the sample could be determined in a liquid scintillation analyser (19OOCA; Packard Instrument Company). The concn of unlabelled cGMP in the samples was then determined by inter- polation from a standard curve.

Acknowledyement- -We would like to thank Dr J. Glossl and Dr L. Mach (Institute of Applied Genetics, Univer- sity of Agriculture, Vienna) for their assistance during the performance of radioimmunoassay.

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