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First published online August 25, 2003

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Nitric oxide mediates seasonal muscle potentiation in clam gills

Louis F. Gainey, Jr^1,* and Michael J. Greenberg²

¹ Department of Biological Sciences, University of Southern Maine, Portland, Maine, 04104, USA
² The Whitney Laboratory, The University of Florida, 9505 Ocean Shore Boulevard, St Augustine, Florida, 32086, USA

View larger version (35K): [in a new window]   Fig. 1. Diagrammatic anatomy of Mercenaria mercenaria (adapted from Gainey et al., 2003). (A) Cross section of a clam. (B) Cross sections through portions of relaxed and contracted demibranchs cut in an anterior-posterior direction, i.e. in and out of the plane of the page with respect to the top figure. (C) Details of a water tube: with the musculature relaxed (left), and contracted (right). The water tube muscles are within the walls of the horizontal blood vessels, the vessels are not shown here. The longitudinal muscles lie between the water tube muscles (and the horizontal blood vessels) and the base of the gill filaments. bv, blood vessel; gf, gill filament; lm, longitudinal muscle; s, septum; wtm, water tube muscle.

View larger version (14K): [in a new window]   Fig. 2. Experimental designs used to construct internal and external contraction ratios. In general, internal contraction ratios were used to ascertain elements of the signaling pathway leading to potentiation e.g. enzyme inhibitors that block potentiation, whereas external contraction ratios were used to ascertain elements that mimic potentiation e.g. DEANO, and to determine if any of the enzymes are involved in the initial response to 5HT. Traces represent contraction of the gill muscles in response to 5-hydroxytryptamine (5HT; upward arrows) followed by flushing of the organ bath and relaxation of the muscle (downward arrows). The enzymes inferred for the signaling pathway are boxed. -, enzyme inhibitors; +, stimulators; hc, height of control contraction; ht, height of treatment contraction; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; L-NAME, a NOS inhibitor; sGC, soluble guanylate cyclase; ODQ, a sGC inhibitor; cGMP, cyclic GMP; PKG, protein kinase G; Rp-8-CPT-cGMPS, a PK-G inihibitor; DEANO, an NO generator; db-cGMP, dibutyryl cGMP, a cell permeable cGMP analog.

View larger version (77K): [in a new window]   Fig. 10. Seasonal variation in the distribution of nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) in the gills of Mercenaria mercenaria. (A) Thick (100 µm) section; muscle fibers visualized with green-fluorescing phalloidin. Muscle is concentrated in the longitudinal muscle (lm), which also sends branches into each gill filament (gf); muscle also occurs in the major blood vessel (bv). (B) The same section as A but visualized with a filter for red fluorescence showing the distribution of immunoreactive (ir) NOS. This section was made in November (early in the season of potentiation) in gills from a Massachusetts clam. (C) Thick section showing the distribution of NOS in a gill taken from a Massachusetts clam in August (no potentiation) 2002. (D) Thin section (12 µm) showing the distribution of NOS in a gill taken from a Florida clam in July (no potentiation) 2001. Note the intense ir-NOS at the base of the gill filaments and in varicose fibers adjacent to the longitudinal muscle. (E) Thick section of a gill in April (potentiation) showing the distribution of ir-sGC, which is concentrated in the longitudinal and septal muscles and the gill filaments. (F) Thick section of a gill in August showing the distribution of ir-sGC, which is concentrated in the gill filaments and, to a lesser extent, in the projections of the longitudinal muscle in the gill filaments. Note the lack of fluorescence in the main body of the longitudinal muscles. All thick sections are 100 µm. hbv, horizontal blood vessel; s, septum; sbv, septal blood vessel; wt, water tube. Scale bars, 50 µm (except for D, 25 µm).

View larger version (6K): [in a new window]   Fig. 3. Contractions of gill muscles in response to 2x10-5 mol l-1 5HT (applied at the upward arrows). The response to each dose of 5HT was terminated by flushing the organ bath (at downward arrows). 1 h elapsed between the flush and the addition of the second dose of 5HT.

View larger version (12K): [in a new window]   Fig. 4. Effect on the internal contraction ratio of the length of time between the first flush of the organ bath and the addition of the second dose of 2x10-5 mol l-1 5HT. Note the scale break between 3 and 24 h. Values are means ± 1 S.E.M., N= 18 (15 min), 98 (30 min), 41 (60 min), 8 (2 h), 4 (3 h), 15 (24 h).

View larger version (13K): [in a new window]   Fig. 5. Monthly variation in a five-year set of mean internal contraction ratios in response to 2x10-5 mol l-1 5HT. Each demibranch was exposed twice to 5HT with a 30 min or 1 h period of relaxation between exposures; the internal contraction ratio is the second response/initial response for each demibranch. Values are means ± 1 S.E.M., N=14 (Jan), 23 (Feb), 51 (Mar), 35 (Apr), 39 (May), 46 (June), 80 (July), 39 (Aug), 32 (Sept), 30 (Oct), 17 (Nov), 13 (Dec).

View larger version (11K): [in a new window]   Fig. 6. The effect of L-NAME on internal contraction ratios. (A) Contractions of the same demibranch in response to 2x10-5 mol l-1 5HT (applied at the upward arrows). The response to each dose of 5HT was terminated by flushing the organ bath (downward arrows). L-NAME (10-4 mol l-1) was applied for 15 min before the second contraction. The resulting internal contraction ratio was 0.92. (B) Various concentrations of L-NAME were applied to isolated demibranchs for 15 min between their first and second contractions in response to 2x10-5 mol l-1 5HT Values are means ± 1 S.E.M., N=3 for all concentrations of L-NAME except 10-4 mol l-1 where N=4.

View larger version (17K): [in a new window]   Fig. 7. The effect of L-NAME on dose-dependent muscle contraction (as a percentage of resting length) in response to 5HT. Treatment demibranchs (solid circles/solid line) were exposed to 1x10-5 mol l-1L-NAME for 15 min, and then both treatment and control demibranchs (open circle/broken line) were exposed to increasing concentrations of 5HT. Data were recorded using ultrasound, which allowed a single demibranch to be used for an entire dose-response curve. Data consist of paired demibranchs (three treatments, three controls) from three clams.

View larger version (12K): [in a new window]   Fig. 8. The effect of L-NAME on mean external contraction ratios. In this experiment, one demibranch was exposed to L-NAME for 15 min prior to exposure to 2x10-5 mol l-1 5HT, while the control demibranch was only exposed to 2x10-5 mol l-1 5HT. The open circle represents data collected in August, when potentiation is absent. Values are means ± 1 S.E.M., N=3-10.

View larger version (11K): [in a new window]   Fig. 9. Mean external contraction ratios in response to an NO donor, DEANO. The treatment demibranchs (numerator) were exposed first to DEANO, and then to 2x10-5 mol l-1 5HT, while the control demibranchs (denominator) were only exposed to 2x10-5 mol l-1 5HT. The open circle represents data collected in July, when potentiation is absent. Values are means ± 1 S.E.M., N=3-15 at each concentration of DEANO.

View larger version (11K): [in a new window]   Fig. 11. Mean external contraction ratios in response to the membrane permeable cGMP analog, dibutyryl cGMP. The treatment demibranchs (numerator) were exposed to dibutryl cGMP and then to 2x10-5 mol l-1 5HT, while the control demibranchs (denominator) were only exposed to 2x10-5 mol l-1 5HT. Values are means ± 1 S.E.M., N=8 for each concentration of dibutryl cGMP.

View larger version (9K): [in a new window]   Fig. 12. Mean internal contraction ratios in response to the membrane permeable guanylate cyclase inhibitor ODQ. ODQ was added between the first and second contractions in response to 2x10-5 mol l-1 5HT. Values are means ± 1 S.E.M., N=6 for each concentration of ODQ.

View larger version (11K): [in a new window]   Fig. 13. Mean external contraction ratios in response to the membrane permeable guanylate cyclase inhibitor ODQ. Treatment demibranchs (numerator) were exposed to 10-6 mol l-1 DEANO and varying concentrations of ODQ and then to 2x10-5 mol l-1 5HT, while the control demibranchs (denominator) were only exposed to 2x10-5 mol l-1 5HT. Values are means ± 1 S.E.M., N=6-4 at each concentration of ODQ.

View larger version (20K): [in a new window]   Fig. 14. Dose-dependent muscle contraction (as a percentage of the resting length) in response to 5HT. Each point is the response of a separate demibranch. Open circles/broken line: data collected in July and August. Solid circle/solid line: data collected between December and May.

View larger version (13K): [in a new window]   Fig. 15. Monthly dependence of the response to 2x10-5 mol l-1 5HT. Means are the response of demibranchs to an initial exposure to 2x10-5 mol l-1 5HT. Values are means ± 1 S.E.M. Sample sizes are the same as Fig. 4.

View larger version (16K): [in a new window]   Fig. 16. Model depicting the signal transduction events leading to potentiation of gill muscle contraction. The coupling between the 5HT receptor is unknown, possibly via a G protein or the receptor being part of a calcium channel. The presence of calcium channels is inferred because the calcium channel blocker verapamil inhibits contraction (L. F. Gainey, personal observation). The existence of a neuronal-like NOS activated by calmodulin is based upon sequence data (L. L. Moroz and B. Untch, personal communication). The protein target(s) of phosphorylation by protein kinase G (PK-G) are also unknown, but based upon data from other molluscs, the target may be an L-type calcium channel, but the 5HT receptors or contractile proteins may also be phosphorylated. -, enzyme inhibitors; + stimulators;?, uncertainty (also broken lines). CaM, calmodulin; nNOS, neuronal-like nitric oxide synthase; NO, nitric oxide; cGMP, cyclic GMP.

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