Long-term hypoxia increases calcium affinity of BK channels in ovine fetal and adult cerebral artery smooth muscle.

Acclimatization to high-altitude, long-term hypoxia (LTH) reportedly alters cerebral artery contraction-relaxation responses associated with changes in K(+) channel activity. We hypothesized that to maintain oxygenation during LTH, basilar arteries (BA) in the ovine adult and near-term fetus would show increased large-conductance Ca(2+) activated potassium (BK) channel activity. We measured BK channel activity, expression, and cell surface distribution by use of patch-clamp electrophysiology, flow cytometry, and confocal microscopy, respectively, in myocytes from normoxic control and LTH adult and near-term fetus BA. Electrophysiological data showed that BK channels in LTH myocytes exhibited 1) lowered Ca(2+) set points, 2) left-shifted activation voltages, and 3) longer dwell times. BK channels in LTH myocytes also appeared to be more dephosphorylated. These differences collectively make LTH BK channels more sensitive to activation. Studies using flow cytometry showed that the LTH fetus exhibited increased BK ß1 subunit surface expression. In addition, in both fetal groups confocal microscopy revealed increased BK channel clustering and colocalization to myocyte lipid rafts. We conclude that increased BK channel activity in LTH BA occurred in association with increased channel affinity for Ca(2+) and left-shifted voltage activation. Increased cerebrovascular BK channel activity may be a mechanism by which LTH adult and near-term fetal sheep can acclimatize to long-term high altitude hypoxia. Our findings suggest that increasing BK channel activity in cerebral myocytes may be a therapeutic target to ameliorate the adverse effects of high altitude in adults or of intrauterine hypoxia in the fetus.

Resting Membrane Potential Modulates Chemoreceptor Sensitivity in Nematocyst Discharge of the Sea Anemone Exaiptasia diaphena.

AbstractExtracellular calcium has been known to be required for in situ nematocyst discharge for more than 60 years, yet calcium's role in nematocyst discharge is poorly understood. Currently, we know that extracellular calcium plays at least two distinct roles in in situ nematocyst discharge. First, calcium plays a role in the triggering of discharge by physical contact, most likely involving transient receptor potential channels. Second, activated L-type calcium channels desensitize nematocyst discharge predisposed to discharge by stimulated chemoreceptors for N-acetylated sugars, such as N-acetylneuraminic acid (NANA). It is not known whether the stimulated NANA signaling pathway activates L-type channels electrogenically through membrane depolarization or directly by phosphorylation of the channel. We hypothesize that the activated NANA signaling pathway initiates desensitization by depolarizing cell membrane potentials to activate voltage-gated L-type calcium channels. Consistent with our hypothesis, we show that depolarization induced by blocking voltage-gated potassium channels with 4-aminopyridine selectively activates Ca2+ influx into tentacle ectodermal cells via L-type channels and inhibits in situ nematocyst discharge from chemosensitized anemones. Furthermore, preventing membrane depolarization with valinomycin or hyperpolarizing resting membrane potentials with low-potassium seawater suppresses NANA-induced Ca2+ influx, prevents desensitization of in situ nematocyst discharge, and enhances NANA sensitivity. Thus, changing resting membrane potentials modulates NANA sensitivity, and NANA-induced depolarization drives desensitization. We suggest that desensitization of the NANA signaling pathway occurs by a feedback pathway involving calcium channels that are activated by NANA-induced depolarization. Elucidating the desensitization pathway may suggest methods to protect or prevent public health cases of nematocyst stinging.

N-Acetyl Neuraminic Acid (NANA) Activates L-Type Calcium Channels on Isolated Tentacle Supporting Cells of the Sea Anemone (Aiptasia pallida).

AbstractSensory receptors control nematocyst discharge on sea anemone tentacles. Micromolar N-acetylated sugars (e.g., N-acetyl neuraminic acid [NANA]) bind chemoreceptors on ectodermal supporting cells and predispose adjacent nematocyst discharge in response to mechanical contact via a cyclic adenosine monophosphate (cAMP)-dependent sensitization pathway, while higher NANA levels dose-dependently desensitize. Recent evidence implicates L-type calcium channels in desensitizing the pathway in aconitate sea anemones Aiptasia pallida (also known as Exaiptasia diaphana). We, therefore, hypothesize that NANA activates calcium influx via L-type calcium channels. We demonstrate a dose-dependent, NANA-activated 45Ca influx into dissociated ectodermal cells isolated from A. pallida tentacles, with maximal influx occurring at desensitizing concentrations of NANA. The L-type calcium channel inhibitors nifedipine, diltiazem, methoxyverapamil, and cadmium blocked NANA-stimulated 45Ca influx. Elevated extracellular KCl levels dose-dependently increased nifedipine-sensitive 45Ca influx to implicate voltage-gated calcium channels. Forskolin, 8-bromo-cAMP, and the protein kinase A inhibitor H-8 affect NANA-stimulated calcium influx in a manner consistent with activated cAMP-dependent pathway involvement. Because NANA chemoreceptors localize to supporting cells of cnidocyte supporting cell complexes, NANA activation of 45Ca influx into isolated tentacle ectodermal cells suggests that L-type calcium channels and NANA chemoreceptors co-localize to supporting cells. Indeed, a fluorescent marker of L-type calcium channels localizes to the apical ectoderm adjacent to nematocysts of live tentacles. We conclude that supporting cell chemoreceptors activate co-localized L-type calcium channels via a cAMP-dependent mechanism in order to initiate desensitization. We suggest that pathway desensitization may conserve nematocysts from excessive discharge during prey capture.

Cnidocyte Supporting Cell Complexes Regulate Nematocyst-Mediated Feeding Behaviors in the Sea Anemone Diadumene lineata.

AbstractCnidarians, as model animals for studying conserved feeding behavior, possess the simplest nervous and digestive systems. Feeding behavior in cnidarians begins with nematocyst-mediated prey retention, proceeds to coordinated tentacle movements and mouth opening, and then proceeds to release of retained prey for ingestion. Understanding the basis of nematocyst discharge, retention, and release is central to explaining cnidarian feeding. Based on studies using artificial targets, cnidocyte supporting cell complexes (CSCCs) regulate nematocyst discharge, retention, and release in Actinaria (sea anemones); but the relevance of CSCCs to prey retention and ingestion has not yet been established. CSCCs exist as three functional types (Types A, B, and C), with a ratio of Types A∶B∶C of 2∶2∶1 in Diadumene lineata (a.k.a. Haliplanella luciae). We tested the hypothesis that CSCCs control nematocyst-mediated prey killing and ingestion. We used a quantitative feeding assay involving Artemia nauplii (prey) and monoclonal D. lineata. The ratios of Types A∶B∶C involved in prey killing and ingestion were 1∶2.5∶5 and 1∶2∶3, respectively. These findings support the CSCC hypothesis. They also indicate that Type Cs predominate in killing small, hard-surfaced, motile, crustaceous prey. Chemoreceptor-bearing Type Bs and Type As assist in prey killing and assume somewhat greater roles in ingestion. Thus, CSCC types differ with respect to their afferent sensory roles as well as their subsequent efferent roles in killing and ingestion. We conclude that CSCC types perform overlapping and complementary roles during feeding.

Activated L-Type Calcium Channels Inhibit Chemosensitized Nematocyst Discharge from Sea Anemone Tentacles.

Because in vivo nematocyst discharge requires extracellular Ca2+, Ca2+ channels have been suspected to be involved; but their identity and role have not been revealed. The majority of nematocysts that discharge from sea anemone tentacles are under the control of sensitizing chemoreceptors for N-acetylated sugars (e.g., N-acetylneuraminic acid). Activated chemoreceptors predispose contact-sensitive mechanoreceptors to trigger discharge. We show that activating L-type Ca2+ channels inhibits N-acetylneuraminic acid-sensitized discharge, contrary to a previous suggestion. In addition, inhibiting L-type channels increases sensitivity to N-acetylneuraminic acid. Specifically, we show that the L-type Ca2+ channel activator (-)-Bay K 8644 dose-dependently inhibits N-acetylneuraminic acid-sensitized discharge, as does raising ambient Ca2+ levels. We also show that lowering extracellular Ca2+ levels or adding any of several selective and chemically distinct L-type Ca2+ channel blockers, including dihydropyridines, dose-dependently increases N-acetylneuraminic acid sensitivity and broadens the dynamic range of N-acetylneuraminic acid sensitization. Consistent with these functional findings, Aiptasia pallida expresses an L-type Ca2+ channel α subunit transcript that encodes a conserved dihydropyridine-binding site. Phylogenetic analysis confirms a close relationship of the Aiptasia Ca2+ channel α subunit sequence between anemones, anthozoans, and cnidarians that extends into protostomal and deuterostomal bilaterians. We conclude that L-type Ca2+ channel activity modulates N-acetylneuraminic acid-sensitized nematocyst discharge in a push-pull manner depending on channel activity state. Our findings suggest that L-type channel activation promotes chemosensory desensitization, and we predict that N-acetylneuraminic acid chemoreceptor signaling will activate L-type channels.

Effects of satiation and starvation on nematocyst discharge, prey killing, and ingestion in two species of sea anemone.

Studies spanning 60 years with several cnidarian species show that satiation inhibits prey capture and ingestion and that starvation increases prey capture and ingestion. Most have attributed the effects of satiation to inhibition of nematocyst discharge. We hypothesized that satiation inhibits prey capture and ingestion in sea anemones (Haliplanella luciae and Aiptasia pallida) primarily by inhibiting the intrinsic adherence (i.e., holding power) of discharging nematocysts. Using a quantitative feeding assay for H. luciae, we found that satiation completely uncoupled prey killing from prey ingestion, while nematocyst-mediated prey killing was only partially inhibited. Using A. pallida to measure nematocyst discharge and nematocyst-mediated adhesive force, we showed that satiation completely inhibited the intrinsic adherence of discharging nematocysts from Type B and Type C cnidocyte/supporting cell complexes (CSCCs), while only partially inhibiting nematocyst discharge from Type Bs. These inhibitory effects of satiation were gradually restored by starvation, reaching a maximum at 72 h after feeding. Thus, the effects of satiation and starvation on prey killing and ingestion in two species of acontiate sea anemones are primarily due to changes in the intrinsic adherence of nematocysts from both Type B and Type C CSCCs.