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N-Acetylneuraminic Acid (NANA) Stimulates in situ cyclic AMP Production in Tentacles of Sea Anemone (Aiptasia pallida): Possible Role in Chemosensitization of Nematocyst Discharge

V. Haktan Ozacmak¹, Glyne U. Thorington¹, William H. Fletcher² and David A. Hessinger^1,*

¹ Department of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA 92354, USA
² Department of Anatomy, School of Medicine, Loma Linda University, Loma Linda, CA 92354, USA

View larger version (30K): [in a new window]   Fig. 1. Diagram of part of an anemone tentacle in cross section (cells not drawn to scale). The tentacle of Aiptasia pallida is composed of two epithelial layers; ectoderm (epidermis) and endoderm (gastrodermis) separated by a basement membrane termed the mesoglea. The ectodermal layer consists of three regions: a monolayer of longitudinal muscle cells closest to the mesoglea; an intermediate layer of neuritic processes and neurons termed the neural plexus; and the compositionally diverse apical epithelial layer, which includes mucus-secreting cells (MC), multicellular complexes known as cnidocyte/supporting cell complexes (CSCCs) and relatively rare sensory cells. The CSCCs consist of a cnidocyte about 35µm in length surrounded by two or more supporting cells (SC). Three different functional types of CSCC have been identified. Two kinds of cnidocyte occur in the tentacle: the nematocyst-containing nematocyte (CN) and the spirocyst-containing spirocyte (SP). The nematocyte sports a single kinocilium from its apical surface which, in turn, associates closely with a bundle of stereocilia contributed from the adjacent supporting cells of the same CSCC. Together, the kinocilium and stereocilia form vibration-sensitive hair bundles. Sensitizing chemoreceptors for N-acetylated sugars, such as N-acetylneuraminic acid (NANA), occur on the apical surfaces of the supporting cells.

View larger version (10K): [in a new window]   Fig. 2. Effects of extraction buffer on the cAMP content of untreated ecto- and endodermal layers from Aiptasia pallida tentacles. Tentacles of sea anemones starved for 72h were individually excised, snap-frozen, stored at -80°C and later extracted in two different media. For measurement of ectodermal cAMP levels, either 1ml of ice-cold 1moll-1 formic acid, pH2.0 (filled columns), or 1ml of ice-cold 0.05moll-1 acetate buffer, pH5.8 (open columns), was added to each frozen tentacle. The cAMP and protein contents of the soluble, freeze-thawed ectodermal layers were assayed. For measurement of endodermal cAMP levels, the insoluble residue, consisting of intact endoderm plus mesoglea, was homogenized in 1ml of 1moll-1 formic acid or 1ml of 0.05moll-1 acetate buffer, pH5.8, on ice, and then assayed for cAMP and protein. Data are expressed as fmolcAMPµg-1protein. Twenty-eight sea anemones were used. Values are means ± S.E.M. (N=2 experiments; n=17 samples).

View larger version (14K): [in a new window]   Fig. 3. Effects of IBMX on tentacle ectodermal cAMP content following treatment with and without N-acetylneuraminic acid (NANA). A group of sea anemones starved for 72h was incubated in filtered, natural sea water, pH7.65, with or without 10-4moll-1 IBMX for 20min (open columns). A second group of sea anemones starved for 72h was incubated in filtered, natural sea water, pH7.65, containing 1.8x10-5moll-1 NANA with or without 10-4moll-1 IBMX for 20min (filled columns). Individual tentacles from each group were snap-frozen, extracted and assayed for ectodermal cAMP using 1moll-1 formic acid. Data are expressed as fmolcAMPµg-1protein. Four anemones were used. Values are means ± S.E.M. (n=8 samples).

View larger version (14K): [in a new window]   Fig. 4. Effects of N-acetylneuraminic acid (NANA) and Mg-ASW on tentacle ectodermal cAMP content. Following 72h of starvation, one group of four anemones was pre-incubated in artificial sea water (ASW) containing 10-4moll-1 IBMX for 10min followed by incubation in ASW containing either 10-4moll-1 IBMX and 1.8x10-5moll-1 NANA (filled columns) or 10-4moll-1 IBMX alone (open columns) for 15min. Individual tentacles from each group were collected, snap-frozen, extracted and assayed for ectodermal cAMP using 1moll-1 formic acid. Data are expressed as fmolcAMPµg-1protein. A second, similar group of four anemones was treated as above except that Mg-ASW replaced ASW. Values are means ± S.E.M. (n=20 samples).

View larger version (22K): [in a new window]   Fig. 5. Effects of N-acetylneuraminic acid (NANA) on nematocyst discharge, adhesive force and tentacle cAMP content. Pre-starved anemones were incubated in filtered, natural sea water containing the specified NANA concentrations for 10min, and in situ adhesive force and nematocyst discharge were measured. Nematocysts on six probes were counted for each dose tested, and the results are the mean of three experiments (, N=3 experiments; n=18 samples). Values of adhesive force measurements were expressed in newtons as the mean of three experiments (, N=3; n=27). For measurement of cAMP, pre-starved anemones were pre-incubated in filtered, natural sea water containing 10-4moll-1 IBMX for 20min followed by incubation in sea water containing 10-4moll-1 IBMX and different concentrations of NANA for 15min. Individual tentacles were excised, snap-frozen and subsequently assayed for cAMP content of both ectoderm (, N=2; n=20) and endoderm (, N=1; n=10) with 1moll-1 formic acid, as in Fig.1. Results are expressed as fmolcAMPµg-1protein. Untreated control values (C) are indicated at the left-hand end of the x-axis. The number of anemones used in each experiment ranged between 16 and 20. Values are means ± S.E.M.

View larger version (15K): [in a new window]   Fig. 6. Time course of N-acetylneuraminic acid (NANA)-stimulated cAMP content of tentacle ectoderm. Pre-starved anemones were pre-incubated in filtered, natural sea water containing 10-4moll-1 IBMX for 20min followed by incubation in filtered, natural sea water containing 10-4moll-1 IBMX and 1.8x10-5moll-1 NANA for specified times. Tentacles were excised, snap-freezen and assayed for ectodermal cAMP content as in Fig.4. Two sets of experiments were combined: one measured the cAMP contents between 0 and 5min of incubation with NANA (, N=3 experiments; n=36 samples) and the other between 5 and 45min of incubation with NANA (, N=3; n=22). The value for 5min of incubation was the average of means from the two sets of experiments. Data are expressed as fmolcAMPµg-1protein. The number of sea anemones used in each experiment ranged between 18 and 20. Values are means ± S.E.M.

View larger version (11K): [in a new window]   Fig. 7. cAMP-dependent protein kinase (PKA) activity in cell-free extracts of tentacles. Tentacles were collected, pooled and homogenized by mortar and pestle under liquid nitrogen followed by sonication for 2–4s in 1ml of ice-cold extraction buffer, pH6.8. The homogenate was then centrifuged at 20000g for 20min at 4°C. The supernatant was immediately used as the source of enzyme activity. PKA activity was assayed with 35µmoll-1 kemptide in the presence and absence of 0.1mmoll-1 exogenous cAMP using undiluted (filled columns) and 1:1 diluted (open columns) supernatants. The protein content of undiluted supernatants was 176µgml-1. Data are expressed as pmoles of phosphate transferred per minute. Values are means ± S.E.M. (N=2 experiments; n=4 samples).

View larger version (15K): [in a new window]   Fig. 8. Effects of substrate concentration on cAMP-dependent protein kinase (PKA) activity in cell-free extracts of tentacles. Tentacles were pooled and processed for the assay of PKA activity as in Fig.6. The supernatant extract was immediately mixed with different concentrations of kemptide in the reaction mixture in the presence () or absence () of 0.1mmoll-1 cAMP. The protein content of supernatants was 140µgml-1. Data are expressed as pmoles of phosphate transferred per minute. Each data point represents the mean ± S.E.M. (N=2 experiments; n=4 samples).

View larger version (13K): [in a new window]   Fig. 9. Effects of N-acetylneuraminic acid (NANA) on cAMP-dependent protein kinase (PKA) activity in whole-tentacle homogenate. Tentacles were harvested, pooled and gently homogenized on ice in extraction buffer, pH6.8, with 10 strokes of a Dounce all-glass homogenizer. Homogenates including 0.1mmoll-1 GTP and 0.1mmoll-1 ATP were stored on ice for 15min in the presence (filled columns) and absence (open columns) of 1.8x10-5moll-1 NANA, after which 40µl samples were taken for PKA assay at 30°C for 10min. The addition of 20nmoll-1 PKA inhibitor (PKI) completely blocked the PKA activity both in the presence and in the absence of NANA. The protein concentration of the homogenate was 528µgml-1. Data are expressed as pmoles of phosphate transferred per minute. Each data point represents the mean ± S.E.M. (n=4 samples).