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Research report
Inhibitory effect of cyclic guanosine 3
9
,5
9
-monophosphate (cGMP)
on the afferent resting activity in the cephalopod statocyst
a,b a,c ,
*
Yijun Tu
, Bernd U. Budelmann
a
The Marine Biomedical Institute, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1163, USA b
Department of Anatomy and Neurosciences, The University of Texas Medical Branch, Galveston, TX 77555-1043, USA c
Department of Otolaryngology, The University of Texas Medical Branch, Galveston, TX 77555-1063, USA
Accepted 2 August 2000
Abstract
The effects of exo- and endogenous cGMP on the resting activity (RA) of afferent crista fibers were studied in isolated preparations of the statocysts of the cuttlefish Sepia officinalis and the squid Sepioteuthis lessoniana. Bath application of the membrane-permeable cGMP
2
analogs 8-bromo-cGMP (B-cGMP) and N ,29-o-dibutyryl 39,59-cyclic guanosine monophosphate (dB-cGMP), and of the selective inhibitor of cGMP-phosphodiesterase zaprinast (ZAP), caused an inhibition of RA. The inhibitory effects of B-cGMP and dB-cGMP remained when the preparation was pre-treated with: (i) the guanylate cyclase inhibitors 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) or cystamine (CYS); (ii) the adenylate cyclase inhibitors nicotinic acid (NIC-A), 29,39dideoxyadenosine (DDA), or MDL-12330A (MDL); (iii) the guanylate cyclase inhibitor methylene blue (M-BLU) and the adenylate cyclase inhibitor MDL combined; or (iv) the
G G
nitric oxide (NO) synthase inhibitors N -nitric-L-arginine methyl ester HCl (L-NAME) or N -nitro-L-arginine (L-NOARG). These data
indicate that cGMP, as an intracellular messenger, has a tonic inhibitory effect on the RA of afferent crista fibers in cephalopod statocysts.
2000 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters, and receptors
Topic: Second messengers and phosphorylation
Keywords: Nitric oxide; cAMP; Hair cell; Vestibular; Equilibrium receptor organ; Invertebrate
1. Introduction have been described in cephalopod statocysts [25,26]. The
purpose of the present experiments is to investigate the The gas nitric oxide (NO) is an important messenger direct effects of cGMP on the resting activity (RA) in molecule that, via a NO-cGMP signal transduction path- those organs.
way, is involved in intra- and inter-cellular communica- This paper is the third of a series of papers that cover a tion. In most cases, NO mediates its biological effects by step-by-step analysis of the physiological effects of L -activating guanylate cyclase and increasing cGMP syn- arginine–NO–cGMP on the RA of crista afferent fibers thesis. A NO-cGMP signal transduction pathway exists in and are the first comprehensive physiological report at vertebrate as well as in invertebrate tissues and its function organ level for any invertebrate or vertebrate equilibrium
has been considered evolutionarily stable (for references, receptor system. Concomitantly they demonstrate the high
see Refs. [25,26]). value of the cephalopod statocysts as invertebrate model
Whereas much is known about NO and a NO-cGMP systems for vertebrate vestibular and pharmacological signal transduction pathway in vertebrate tissues, compara- research (e.g. Refs. [24–26,29]).
tively little is known in invertebrates (for references, see Refs. [25,26]). Only recently an inhibitory L-arginine /
NO-cGMP and an excitatory L-arginine / NO-cAMP pathway 2. Materials and methods
Experiments were done with 82 isolated statocyst prepa-*Corresponding author. Fax:11-409-772-6993.
E-mail address: bubudelm@utmb.edu (B.U. Budelmann). rations of 36 cuttlefish (Sepia officinalis, males and
females, mantle lengths 131–264 mm) and six squid 3. Results (Sepioteuthis lessoniana, males and females, mantle
lengths 174–268 mm). The cuttlefish and squid were All preparations showed RA, with the majority repre-cultured in the National Resource Center for Cephalopods senting a multi-unit activity. When single units could be at the University of Texas Medical Branch in Galveston. identified, they had a mostly irregular discharge pattern. For a detailed description of the statocyst preparation, as Between preparations, the amplitudes of the potentials well as the experimental design and conditions, see Refs. differed considerably in size, ranging from 5 to 50 mV. [25,26]. In brief, the resting activity (RA; defined as the Because the large potentials were not very stable over activity of the preparation at rest) of the angular accelera- time, most analyses were done with potentials between 6 tion receptor system (crista / cupula system) of the and 12 mV.
statocysts was recorded from axons of the first-order A total of 88 tests were performed with 82 statocysts of afferent units in the large crista nerve (N. crista major). For 42 animals. In 81 tests (92%), applications of the follow-the cellular organization of follow-the crista, see Ref. [3] (for a ing drugs caused inhibitory effects: the membrane-perme-short summary, see Ref. [25]). able cGMP analogs B-cGMP and dB-cGMP; the selective The effects of bath applications of the following drugs cGMP-phosphodiesterase inhibitor ZAP; B-cGMP after on RA were tested: the membrane-permeable cGMP prior applications of the guanylate cyclase inhibitors CYS,
2
analogs 8-bromo-cGMP (B-cGMP) (from RBI) and N ,29- ODQ, and M-BLU; B-cGMP after prior applications of the
o-dibutyryl 39,59-cyclic guanosine monophosphate (dB- adenylate cyclase inhibitors NIC-A, DDA, and MDL; dB-cGMP) (from Sigma); the selective inhibitor of cGMP- cGMP after prior combined applications of the adenylate phosphodiesterase 1,4-dihydro-5-[2-propoxyphenyl]-7H- cyclase inhibitor MDL and the guanylate cyclase inhibitor 1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) (ZAP) M-BLU; and dB-cGMP after prior applications of the NO (from Sigma); the guanylate cyclase inhibitors methylene synthase inhibitorL-NAME andL-NOARG. Only in seven blue (M-BLU) (from RBI), cystamine (CYS) (from tests (8%), the application of a drug had no effect; in five Sigma), and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one of those seven tests, the concentration of the drug quite (ODQ) (from Tocris); the adenylate cyclase inhibitors likely was below threshold.
nicotinic acid (NIC-A) (from Sigma), 29,39-dideoxy- For details of the concentration of the drugs applied and adenosine (DDA) (from Sigma), and N-(cis-2-phenyl- the amount of inhibition (percentage of decrease) of RA cyclopentyl) azacyclotridecan-2-imine-hydrochloride caused by the drugs, see Table 1 and Fig. 1.
(MDL-12,330A) (MDL) (from RBI); and the NO synthase G
inhibitors N -nitric-L-arginine methyl ester HCl (L -G
NAME) and N -Nitro-L-arginine (L-NOARG) (both from 4. Discussion Sigma).
The concentrations given in Section 3 are the con- In the present experiments, the statocysts of the cuttlefi-centrations of the applied test solutions; in all cases, this sh Sepia officinalis and the squid Sepioteuthis lessoniana was 10 ml. Since the statocyst cavity had a volume were very sensitive to bath applications of B-cGMP and between 50 and 100ml, the effective concentration was up dB-cGMP, which are membrane permeable analogs of to an order of magnitude lower (and decreased even further cGMP [12,23]. Both cGMP analogs decreased RA, which with diffusion of the drug into the 10 ml bath solution of suggests that cGMP also has an inhibitory effect on RA.
the dish). Under the experimental conditions used, the threshold
26
Controls: To exclude non-specific actions of the drugs concentration of B-cGMP was at least 10 M and of 28
on RA, the effects of NaCl solutions (amount, pH and dB-cGMP 10 M; most likely, however, each is an order concentrations as for test solutions) and iso-pH seawater of magnitude lower (see Section 2). Application of ZAP (a were tested. No significant effects on RA by any of these selective cGMP-phosphodiesterase type V inhibitor, which
solutions were seen. causes accumulation of endogenous cGMP [27]), also
To analyze the effects of the drugs on RA, a pre- and reduced RA. As to be expected, applications of the cGMP post-stimulus histogram was used and the average levels of analogs remained effective when the preparations were RA before and during the whole period after the drug pre-treated with NO synthase or guanylate cyclase in-application that was above or below the pre-stimulus level hibitors, because in the intracellular signal transduction of RA were compared. To allow comparison and averaging pathway the effect of cGMP is after that of NO synthase of data from different preparations (with different levels of and guanylate cyclase. Applications of the cGMP analogs RA), the pre-stimulus level of RA was used as reference also remained effective when pre-treatment was with level (5100%) and post-stimulus changes .5% were adenylate cyclase inhibitors, or guanylate and adenylate expressed as percentage changes relative to the pre- cyclase inhibitors combined, showing that adenylate stimulus level (2% for decrease, 1% for increase); post- cyclase is not essential for the action of cGMP.
Table 1
Summary of extracellular responses of RA of afferent crista fibers in the statocysts of the cuttlefish Sepia officinalis and the squid Sepioteuthis lessoniana to bath application of various drugs
Substance Concentration No. of No. of % decrease No. of RA
(M) tests RA decrease 6% S.E.M. no change
Sepia officinalis
protein kinases, cGMP-gated ion channel protein kinases, [11,22] whereas the soluble guanylate cyclase functions as and phosphodiesterases; these respectively mediate protein the receptor for NO and nitrovasodilator drugs [30] and phosphorylation, ions across the plasma membrane, and participates in the regulation of blood flow and supporting cyclic nucleotide catabolism [5,10,20]. Presumably across cell physiology [22].
equivalent tissues of the vertebrate inner ear [11,22]. cGMP is known to be directly or indirectly involved in cholinergic signal transmission (for references, see Refs. [17,28]). In cephalopods statocysts, acetylcholine is pres-ent in the efferpres-ent fiber system and has a powerful, mostly inhibitory, action via muscarinic and nicotinic cholinergic receptors [1,29]. It is possible, therefore, that in the present experiments an application of the cGMP analogs mimicked an activation of the cholinergic system. However, the assumed tonic increase of endogenous cGMP (caused by ZAP) cannot be explained by a tonic activity of the cholinergic efferent system because in the preparations used the efferent system was non-functional (the statocysts were cut off the brain) and, therefore, the source for the increase of endogenous cGMP must be another than the cholinergic system; a possibility is the receptor hair cells. In cephalopods, a NO / nitric oxide synthase (NOS) system has been shown for the brain and other nervous tissues [4,9,15], for photoreceptor cells [8], and for the ink gland [16]; NO is also known to be involved in visual and tactile learning [18,19]. For more details about the effects of L-arginine and NO donors on the RA of, and the significance of the L-arginine / NO-cGMP and L-arginine / NO-cAMP pathways in cephalopod statocysts, see discus-sions in Refs. [25,26].
Acknowledgements
This work was supported by NSF grant IBN 96-03693 (B.U.B.). The animals were obtained from the National Resource Center for Cephalopods of the Marine Bio-medical Institute of the University of Texas Medical Branch at Galveston. The authors thank Andrew F. Bur-rows for his help with, and improvement of, some of the computer programs.
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