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Pharmacological Disruption of Sea Urchin Tube Foot Motility and Behavior.

Introduction

The deuterostome superphylum comprises the chordates, which include all vertebrates, hemichordates (acorn worms), and echinoderms (Satoh et al., 2014); echinoderms themselves are comprised of sea urchins, sea stars, brittle stars, sea cucumbers, sand dollars, and crinoids. Sea urchin larvae have been used as model organisms in reproductive and developmental biology for many years, in part due to the ease of obtaining gametes and monitoring the early stages of fertilization and development in the transparent larvae (McClay, 2011). In comparison, much less is known about the cell biology and molecular physiology of adult sea urchins and how they compare to other animal systems. Following metamorphosis from larvae, sea urchins exhibit pentaradial symmetry and develop a hard calcified test from which protrude numerous spines and tube feet. Within the test is the gastrointestinal tract, male or female gonads, a nervous system, and a water vascular system that controls tube feet relaxation and extension (Nichols, 1966). The disk regions of echinoderm tube feet contain cells that secrete adhesive and de-adhesive substances to enable the attachment and detachment of feet to surfaces (Flammang et al., 1998; Santos et al., 2009). All adult sea urchins are benthic, residing with their oral surface facing the substrate, and they translocate by movement of the highly mobile tube feet. One behavior that has been extensively studied in sea urchins is the righting response, in which an inverted sea urchin will use its tube feet and spines to reorientate itself back to the upright (oral-down) position (Reese, 1966; Sherman, 2015). Sea urchins can also detect light despite lacking eyes (Millott, 1954), and there is evidence that sea urchins have low-resolution vision (Blevins and Johnson, 2004; Ullrich-Luter et al., 2011; Yerramilli and Johnsen, 2011). Light detection is mediated by a system of photoreceptors localized within the terminal disk regions of tube feet, within spines, and across the test (Ullrich-Luter et al., 2011, 2013). Behaviorally, light detection is apparent in the negative phototaxic responses of many sea urchin species (Yoshida, 1966; Adams et al., 2001).

Although sea urchins can sense and respond to their environment and have nervous tissue, they lack many of the nervous system features that are found in almost all other animals. Adult sea urchins, unlike their larvae, have a decentralized nervous system (Cobb, 1985; Burke et al., 2006; Burke, 2011), lacking a brain or any centralized enlarged ganglia common to most other animals. The sea urchin nervous system consists of a peripharyngeal nerve ring that encircles the esophagus close to the oral cavity within the Aristotle's lantern. From this ring radiate nerve cords that connect to the subepithelial nerve net that innervates tube feet and spines (Florey et al., 1975; Smith et al., 1985; Burke et al., 2006).

The sequenced genome of the purple sea urchin, Strongylocentrotus purpuratus, revealed many genes that would be expected to be involved in sea urchin sensory perception, nervous system function, and behavioral responses (Burke et al., 2006). Here we adopted a pharmacological approach to disrupt sea urchin behavior as a first step in identifying the molecular components responsible for sea urchin sensory and motor function. We first investigated the suitability of different methods of drug administration in disrupting sea urchin behavior by using the righting response as an assay for drug delivery. We then attempted to disrupt sea urchin phototaxis using drugs that would be expected to inhibit known components of the light signal transduction from other animals. Because it is unknown whether the light signal transduction pathway in sea urchins is more similar to that found in other invertebrates such as arthropods or to that in vertebrates (Yarfitz and Hurley, 1994), we attempted to inhibit the negative phototaxis of the sea urchins by separately inhibiting transient receptor potential (TRP) channels, a key component in Drosophila light signal transduction (Minke and Selinger, 1996), and by inhibiting phosphodiesterase (PDE) activity, a key component of light signal transduction in vertebrates (Takemoto and Cunnick, 1990). Because both drugs were found to inhibit movement and the righting response, and it is known that both TRP channels and PDE play a role in smooth muscle function in Drosophila and mammals (Rybalkin et al., 2003; Earley and Brayden, 2015), we next determined the effects of inhibiting extracellular [Ca.sup.2+] entry and nitric oxide (NO) synthase on the righting response, because both would also be expected to inhibit smooth muscle activity, based on known mechanisms studied in other animals (Vials and Burnstock, 1992).

Materials and Methods

Animals

Purple sea urchins, Strongyiocentrotus purpuratus (Stimpson, 1857), about 60 mm in test diameter, from the Pacific Ocean were obtained from Marinus Scientific (Long Beach, CA) and maintained in aerated tanks at 13 [degrees]C containing artificial seawater (ASW) (Instant Ocean; That Pet Place, Lancaster, PA) made according to the manufacturer's instructions. Variegated sea urchins, Lytechinus variegatus (Lamarck, 1816), from the Gulf of Mexico were obtained from Gulf Specimen Marine Laboratories (Panacea, FL) and were maintained at 20 [degrees]C in aerated tanks containing ASW.

The major ion concentrations in Instant Ocean have been measured (in mmol [l-.sup.1]) as [Na.sup.+] (462), [K.sup.+] (9.4), [Mg.sup.2+] (52), [Ca.sup.2+] (9.4), CF (550), S[O.sup.2-.sub.4] (28), and [Li.sup.+] (20) (Atkinson and Bingman, 1997). For [Ca.sup.2+]-free experiments, ASW was used consisting of (in mmol [l-.sup.1]) NaCl (437), KCl (9), Mg[Cl.sub.2] 6[H.sub.2]0 (22.9), MgS[O.sub.4] 7[H.sub.2]O (25.5), and NaHC[O.sub.3] (2.1), pH adjusted to 7.5. All behavioral tests were conducted in ASW solutions chilled to 10-15 [degrees]C.

To identify individual animals, each sea urchin was removed from ASW, and one to three colored beads were attached to spines using superglue. Once the glue had dried, the sea urchins were returned to ASW tanks.

Drug administration

All drugs were purchased from Sigma-Aldrich (St. Louis, MO). The following drug stock solutions were made and used immediately or stored at -20 [degrees]C until required: 805 mmol [l.sup.-1] hexamethonium in water, 400 mmol [l.sup.-1] 2-aminoethoxydiphenyl borate (2-APB) in methanol, 10 mmol [l.sup.-1] diltiazem in water, 1 mmol [l.sup.-1] thapsigargin in dimethyl sulfoxide (DMSO), 10 mmol [l.sup.-1] N(G)-nitro- L-arginine methyl ester (L-NAME) in ASW, and 100 mmol [l.sup.-1] theophylline in water. A few drops of concentrated ammonium hydroxide were added to the theophylline stock solution to solubilize the theophylline. Once the theophylline completely dissolved, the pH was reduced to 7.5 using HCl. Test solutions were made fresh each day from stocks and diluted in ASW.

A variety of different drug administration procedures were tested. For most immersion tests, sea urchins were immersed in small tanks containing 750 ml ASW for 1 h, and the behavioral tests were conducted in the drug solutions. Some behavioral tests were conducted in ASW only, following immersion of the sea urchins in drug solution for 15 minutes.

For oral administration, sea urchins were removed from the ASW, and a 22-gauge needle and syringe were used to pry open the sea urchin mouth and apply the drug solution directly to the digestive tract. For administration by intra-coelomic injection, sea urchins were removed from the ASW, a 22-gauge needle was inserted into the soft tissue around the mouth, and 3 ml control or drug solution was slowly applied. For topical application, sea urchins were removed from the ASW, and a foam brush was used to apply drug solution over the entire test. Following either oral, injection, or topical application of drugs, sea urchins were returned to the ASW solution after 15 minutes to allow for drug absorption.

Behavioral assays

To test whether applied compounds affected the sea urchin's ability to move, a righting assay was used. The sea urchins were inverted and placed on their aboral side on the base of a tank containing ASW with or without the specified drugs and modifications, and the time to completely right themselves was recorded. Healthy, untreated sea urchins typically right themselves in about three minutes. A cutoff of 15 minutes was applied to the righting experiments, and for statistical analysis, this cutoff value was used as the time to right. If sea urchins in control conditions failed to right within 15 minutes, they were excluded from further testing for that day. Those sea urchins that did right within 15 minutes in control conditions were subsequently used in drug righting experiments, typically within an hour of the initial control experiment. Sea urchins were allowed to recover for at least two days between chemical exposures.

For each phototaxis assay, a single sea urchin was submerged in an upright (oral-surface-down) orientation in the center of a narrow tank measuring 165 mm x 315 mm x 210 mm. The tank contained either control ASW or ASW plus the stated drug. Light was blocked from the tank on all sides using black cardboard and black tape, except for a 50-mm slit on the lower half of one side, next to which a standard white light filament bulb was placed. An opaque lid was placed on the tank, and the position of the sea urchin after 15 minutes was recorded. Positive values indicate movement of the sea urchin away from the light source. For the dark conditions, the entire tank was blacked out, the light source was removed, and the sea urchin position was recorded after 15 minutes.

To obtain the absorbance spectrum of released pigment, a sea urchin was immersed in 500 [micro]mol [l.sup.-1] 2-APB. After 1 hour, triplicate samples of the ASW were taken, and an absorption spectrum from 220 to 900 nm was obtained using quartz cuvettes blanked against ASW containing 500 [micro]mol [l.sup.-1] 2-APB in an Ultrospec 2100 Pro UV-Visible Spectrophotometer (Biochrom, Cambridge, UK) in wavelength scanning mode at 30 nm [s.sup.-1].

All t tests were two-tailed paired tests and assumed non-equivalent variance.

Results

Drug administration

Our initial aim was to elucidate components of the molecular pathways involved in the sea urchin phototaxic response by using a pharmacological approach. Because there is little information currently available on effective methods of drug administration in sea urchins, we first sought to develop an effective drug administration procedure by using a righting assay. Upon inversion, a healthy sea urchin will right itself within a few minutes (mean = 189 [+ or -] 23 s, n = 10), and this behavior is independent of ambient light (Fig. 1). Injection of the well-characterized muscle nicotinic acetylcholine receptor antagonist hexamethonium at concentrations of millimoles per liter through the soft tissue surrounding the mouth and into the coelom led to a clear decrease in the tone of the tube feet and significantly slowed righting time about fivefold (Fig. 2A, B; Videos 1-3, available online). However, it was also found that control injections of ASW without hexamethonium also led to an approximately twofold significant increase in righting time (Fig. 2C), indicating that the injection procedure itself affected normal sea urchin behavior. We thus sought a less behaviorally disruptive method of drug application. Oral administration, by direct application of hexamethonium solution to the digestive tract via the mouth, was found to have no effect on righting time (Fig. 2D), while topical application via a brush on sea urchins removed from ASW gave variable results, with some sea urchins being completely paralyzed and others showing no obvious effects (data not shown). Immersing in hexamethonium solution for 15 minutes, and then conducting righting experiments in ASW without any drug, was found to be ineffective, presumably due to rapid washout of the drug. However, immersion of the sea urchin in hexamethonium solution was found to be both dose dependent and effective at inhibiting the righting response when the immersion was for at least one hour (Fig. 2E). Returning sea urchins to ASW lacking hexamethonium for several hours led to a recovery of tube feet movement and the righting response.

Targeting potential components of light signal transduction

A phototaxis assay tank was set up as shown (Fig. 3). The setup provided an approximately 20-fold difference in the illuminance between the light-distal and light-proximal ends of the tank, ranging from 2 to 47 kilolux (Fig. 3D). However, it is estimated that the illuminance differential experienced by a sea urchin is actually amplified to an approximately 140-fold difference when a sea urchin is placed in the tank, due to the sea urchin itself casting a shadow over its distal side (Fig. 3E). Using this phototaxis setup, we determined that Strongylocentrotus purpuratus translocated in the opposite direction from the light source, with little movement in the absence of light (Fig. 3B), confirming previous studies showing that S. purpuratus is negatively phototaxic (Giese and Farmanfarmaian, 1963; Ullrich-Luter et al., 2011).

Sea urchins are invertebrates, but within the same superphylum as vertebrates, and it is therefore unknown whether the light signal transduction pathway in sea urchins is more similar to that found in other invertebrates such as arthropods or to that found in vertebrates. We therefore attempted to inhibit the negative phototaxis of the sea urchins by separately inhibiting TRP channels, a key component in Drosophila light signal transduction, and PDE activity, a key component of light signal transduction in vertebrates. Surprisingly, both the TRP channel modulator 2-APB and the PDE inhibitor theophylline were found to inhibit movement of the sea urchins (Fig. 4). The lack of movement following administration of these drugs could be due to disruption of light detection or disruption of movement with no effect on light detection. To determine whether locomotor activity alone was negatively affected by 2-APB and theophylline, we performed righting response time assays on sea urchins in the two drugs. Both 2-APB and theophylline inhibited the righting response in a dose-dependent manner (Fig. 5). Furthermore, when sea urchins were immersed in a high concentration of 2-APB (500 [micro]mol [l.sup.-1]), not only was movement completely inhibited but also there was a visible and dramatic release of pigment from the sea urchins into the ASW (Fig. 5C).

The 2-APB used in these experiments was made by dissolving 2-APB in methanol to a concentration of 400 mmol [l.sup.-1] and further diluting in ASW to give the final experimental 2-APB concentrations. This equates to 0.125% methanol remaining in the 500 [micro]mol F 2-APB experimental solution. Control experiments showed that there was no release of pigment when urchins were immersed in a 0.125% methanol in ASW solution, nor was there any significant effect on righting times (control ASW mean = 221 [+ or -] 53 s, methanol ASW mean = 326 [+ or -] 119 s, paired Student's t test, P = 0.46, n = 4).

Analysis of the absorbance spectrum of this pigmented ASW demonstrated absorbances from 220 to 650 nm, with peaks at about 262, 302, 386, 428, 485, 510, and 542 nm (Fig. 5D). We sought to understand whether the release of pigment is a general response of sea urchins to high concentrations of 2-APB, so we repeated the experiment with a second species, Lytechinus variegatus. There was no visible release of pigment when L. variegatus (n = 2) were immersed in 500 [micro]mol [l.sup.-1] 2-APB for 1 h. High concentrations of 2-APB could potentially lead to overactivation of the visual transduction pathway and the expulsion of the sea urchin's purple pigment as part of a stress response. We therefore sought to determine whether extreme bright light would also lead to a release of pigment as part of this potential stress response. Illumination of S. purpuratus sea urchins with a high-intensity LED flashlight, exposing the aboral surface to illumination of about 45,000 lx, did not lead to visible pigment release, indicating that pigment release was specific to 2-APB application and was not a general feature of potential overactivation of the light detection system.

In vertebrates, both PDE and TRP channels are present in vascular smooth muscle cells and via separate pathways can modulate the contractility of these cells. In both cases, the pathways require the influx of external [Ca.sup.2+]. We found that external [Ca.sup.2+] was essential for normal righting, because in [Ca.sup.2+]-free ASW. sea urchins were completely unable to right themselves. However, doubling the external [Ca.sup.2+], from 9.3 mmol [l.sup.-1] (control) to 18.6 mmol [l.sup.-1], did not significantly affect the control righting time (Fig. 6). The route of entry of [Ca.sup.2+] into the sea urchin contractile cells could be through a variety of different ion channels or transporters located within the cell surface membrane or from intracellular stores. Consistent with [Ca.sup.2+] entry through cell surface calcium channels, the calcium channel antagonist D-cis diltiazem also significantly increased righting time (Fig. 6C), whereas thapsigargin, which acts to deplete intracellular [Ca.sup.2+] stores, had no effect on righting time (Fig. 6D). The thapsigargin used in these experiments was made by dissolving thapsigargin in DMSO to a concentration of 1 mmol 1 and further diluting in ASW to give the final experimental thapsigargin concentrations. This equates to 0.0001% DMSO remaining in the highest thapsigargin concentration used (100 nmol [l.sup.-1]). Control experiments showed that there was no significant effect of DMSO on righting times (control ASW mean = 291 [+ or -] 110 s, 0.0001 % DMSO ASW mean = 279 [+ or -] 78 s, paired Student's t test, P = 0.79, n = 5).

The initiation of relaxation in vertebrate vascular smooth muscle can occur via the synthesis of the well-characterized vasorelaxant NO, which is released from neighboring endothelial cells adjacent to vascular smooth muscle cells. The NO synthase inhibitor L-NAME increased sea urchin righting time at 1 mmol [l.sup.-1] but had no detectable effect on righting time at 0.2 mmol [l.sup.-1], indicating a role of NO in tube foot motility (Fig. 7).

Discussion

Sea urchins have a range of behavioral responses that can be investigated pharmacologically to determine the role of specific enzymes and channels in behavioral processes. Using the muscle nicotinic acetylcholine receptor antagonist hexamethonium, we found that the most effective method of drug administration for behavioral experiments was the immersion of sea urchins in drug solution for an hour, coupled with performing the behavioral experiments in the drug solution. Hexamethonium was used at high concentrations (mmol [l.sup.-1]) to ensure that behavioral effects could be unambiguously observed in the absence of any available pharmacodynamics and pharmacokinetic data of hexamethonium on sea urchin systems. The normal inhibitory range of hexamethonium in other systems is about 1-200 [micro]mol [l.sup.-1] (Harry, 1963; Nooney, 1992).

We demonstrated that intracoelomic injection is an effective drug delivery route; previously it has been shown that compared to immersion, intracoelomic injection results in faster absorption and slower removal of the antimicrobial enrofloxacin in Strongylocentrotus droebachiensis (Phillips et al., 2016). However, our control injection experiments demonstrated that the injection procedure itself can influence sea urchin behavior, and, thus, we continued with the less invasive administration method of immersion.

Sea urchin tube feet are known to contain muscles that contract in response to acetylcholine, and contraction of isolated tube feet can be inhibited by the application of nicotinic acetylcholine antagonists such as hexamethonium and decamethonium (Florey et al., 1975). We demonstrated that antagonizing nicotinic acetylcholine receptor-mediated neurotransmission with hexamethonium could effectively inhibit the righting response, and, thus, the righting response is a rapid, convenient, and reproducible behavior with which to assay sea urchin locomotor responses in whole animals. Nicotinic acetylcholine receptors have also been implicated in the activation of muscles that control sea urchin spine movement (Morales et al., 1989), and spine movement is also an integral feature of the righting response. The S. purpuratus genome contains at least 12 nicotinic acetylcholine receptor subunit encoding genes and multiple acetylcholinesterase genes (Burke et al., 2006); and, therefore, the use of subunit-specific inhibitors (Papke et al., 2005) or positive allosteric modulators (Marotta et al., 2014) may enable the identification of specific subunits involved in locomotor activity.

We found that drugs that inhibited movement in response to light also disrupted the righting response, suggesting that the lack of movement in response to light is due to inhibition of locomotion rather than any specific effect on light detection. We therefore focused our attention on essential molecular components of sea urchin locomotor responses by quantifying the effect of inhibitors on the righting response. We identified multiple molecular components of tube foot muscle activity using pharmacological agents. Compounds that inhibit [Ca.sup.2+] channels, PDE, NO synthase, and TRP channels all inhibited the righting response. The well-characterized [Ca.sup.2+] channel antagonist D-cis diltiazem inhibited sea urchin righting at 500 [micro]mol [l.sup.-1], a concentration that is expected to completely inhibit L-type [Ca.sup.2+] channel activity (McDonald et al., 1994; Hagiwara et al., 1997), indicating a role for [Ca.sup.2+] in the righting response and therefore tube foot function. We also showed that extracellular [Ca.sup.2+] is needed for the righting response, adding further evidence for the role of [Ca.sup.2+] in tube foot function. Thus, like muscles found in all other animals, [Ca.sup.2+] plays a key role in muscle contraction. We also demonstrated that the sarco/endoplasmic reticulum [Ca.sup.2+]-ATPase (SERCA) inhibitor thapsigargin, up to 100 nmol [l.sup.-1], did not affect the righting time. Inhibition of SERCA would be expected to deplete intracellular [Ca.sup.2+] stores, and, thus, these stores may not be essential for the righting response. Although thapsigargin has been shown to inhibit SERCA activity completely at concentrations below 100 nmol [l.sup.-1] (Lytton et al., 1991) in isolated preparations, we cannot rule out that concentrations greater than 100 nmol F may have effects on the righting response or that [Ca.sup.2+] release is required for tube foot function via a different intracellular release mechanism, such as through inositol trisphosphate ([I[P.sub.3]) receptors.

PDE enzymes have well-known roles in the control of smooth muscle tone (Rybalkin et al., 2003) in many organisms. In sea urchins, PDE has been shown to play a role in sperm motility (Su and Vacquier, 2006), but to our knowledge this study is the first demonstration that the PDE inhibitor theophylline can affect adult sea urchin behavior. Theophylline is a nonspecific PDE inhibitor with a half maximal inhibitory concentration (I[C.sub.50]) for isolated smooth muscle relaxation of about 80-300 [micro]mol [l.sup.-1], depending on the tissue (Rabe et al., 1995). This range is consistent with the range of concentrations and effects that we observed on sea urchin righting time, although slightly higher concentrations were needed to disrupt movement in the phototaxis experiments.

Another molecule with a well-characterized role in modulating smooth muscle tone in many organisms is NO (Ignarro, 1990). NO has been found to be a general muscle relaxant of echinoderms, in both sea stars (Elphick and Melarange, 1998) and sea urchins (Billack et al., 1998); and inhibition of NO synthase with 1 mmol F L-NAME has previously been shown to alter the length of isolated sea urchin tube feet (Billack et al., 1998). It is therefore not surprising that we found that 1 mmol [l.sup.-1] L-NAME can inhibit sea urchin righting, which is a behavioral response that requires coordinated tube foot activity.

The 2-APD dose dependently inhibited both the phototaxis response and the righting response, and at 500 [micro]mol [l.sup.-1] it led to a dramatic release of pigment from S. purpuratus. It seems likely that the principal action of 2-APB on inhibiting the phototaxis response is through the inhibition of locomotion. The drug 2-APB is a broad modulator of TRP channels. In mammals, 2-APB at low concentrations of 1-14 [micro]mol [l.sup.-1] inhibits multiple different subtypes of canonical-type (TRPC) and melastatin-type (TRPM) channels (Hu et al., 2004; Colton and Zhu, 2007), whereas from 34 to 322 [micro]mol [l.sup.-1] it has been found to activate some vanilloid-type TRP (TRPV) channels (Hu et al., 2004). We found that at 500 [micro]mol [l.sup.-1] 2-APB, there was an obvious release of pigment from S. purpuratus, which was not observed when 5-50 [micro]mol [l.sup.-1] 2-APB was used. It is therefore possible that the pigment release seen with 500 [micro]mol [l.sup.-1] 2-APV occurs via TRPV channels and that these channels do not play a major role in the sea urchin righting response. Alternatively, greater than 10 [micro]mol [l.sup.-1] 2-APB has been found to block Orai (calcium release-activated calcium channel) in mammalian cells (Prakriya and Lewis, 2001), and thus, the pigment release may be via these channels in sea urchins. It may be that the pigment release observed is indicative of dysregulated shedding of photoreceptor outer segments. The shedding of these outer segments is a normal feature of many animal vision systems and is coupled to phagocytosis of materials by retinal epithelial cells to regenerate photoreceptor disks (Young, 1967; Mazzoni et al., 2014). Previously, TRP channels have been implicated in early sea urchin development (Buznikov et al., 2010); our data suggest that they also play key roles in locomotion and are involved in pigment release.

Given the whole-animal experimental setup, it is unknown whether all of the targets of the drugs that were found to inhibit the righting response reside within a single cell type or tissue. However, intriguingly, [Ca.sup.2+] channels, PDE, NO synthase, and TRP channels have been demonstrated to be involved in modulating vascular smooth muscle tone in vertebrates (Rybalkin et al., 2003; Earley and Brayden, 2015), raising the possibility that similar muscle signal transduction pathways may exist in the water vascular system of sea urchins to control tube foot muscle tone.

The close phylogenetic relationship between echinoderms and vertebrates, the published S. purpuratus genome, and the ease of whole-animal behavioral studies make the underutilized mature sea urchin an attractive candidate as a model organism in neurobiology. In addition, the decentralized nervous system found in many echinoderms may yield insights into the general principles governing computation in diffuse neural systems to regulate complex behavior.

Acknowledgments

This work was supported by funding from Franklin and Marshall College (Faculty Research Grant to CS, Eyler Summer Scholarships to MAS and LMK, and Independent Study Awards to MAS and PJS). In addition, we thank Professor Ryan Lacy for supplying some of the Strongylocentrotus purpuratus specimens used in these studies.

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MUNEEB A. SHAH, LUCY M. KIRKMAN, PHILIP J. SITVER, AND CHRIS SHELLEY (*)

Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania 17604

(*) To whom correspondence should be addressed. E-mail: chrisovertheatlantic@gmail.com.

Abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; DMSO. dimethyl sulfoxide; L-NAME, N(G)-nitro-L-arginine methyl ester; NO, nitric oxide; PDE, phosphodiesterase; SERCA, sarco/endoplasmic reticulum [Ca.sup.2+]-ATPase; TRP, transient receptor potential: TRPC, canonical-type TRP; TRPM, melastatin-type TRP; TRPV. vanilloid-type TRP.

Online enhancements: videos.

Received 3 August 2017; Accepted 6 February 2018; Published online 11 April 2018.
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Author:Shah, Muneeb A.; Kirkman, Lucy M.; Sitver, Philip J.; Shelley, Chris
Publication:The Biological Bulletin
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Date:Apr 1, 2018
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