OBJECTIVE:Here we report initial observations and bioinformatics analysis suggesting that the sea anemone Diadumene lineata can be induced to age when cultured at 16oC. Inducible aging may make D. lineata a useful experimental model for aging studies. This research stemmed from a published study of D. lineata that did not involve or report on inducible aging. RESULTS:Anemones cultured at 16oC displayed several signs of aging not evident when cultured at room temperature (21oC), including the cessation of binary fission, growth to a much larger size, a progressive reduction in the consumption of prey, and ultimately, shriveling. Re-analysis of published RNA-seq data identified 2,824 differentially expressed putative genes in anemones cultured at 16oC vs. room temperature. Functional enrichment analysis of the differentially expressed genes is consistent with an aging phenotype at 16oC, and suggests that the 16oC anemones may undergo gametogenesis prior to the development of senescence. A significant increase in somatic single nucleotide polymorphisms also occurred at 16oC.

Integrated chemo- and mechanosensory pathways, along with activated calcium influxes, regulate nematocyst discharge from sea anemone tentacles. Discharge from vibration-sensitive Type A cnidocyte supporting cell complexes use calcium-conducting transient receptor potential V4-like channels. Because calcium influxes often couple with calcium-activated, large-conductance potassium (BK) channels, we hypothesized that BK channels function in nematocyst discharge. To verify this hypothesis, we first tested five selective BK channel blockers on nematocyst-mediated prey killing in Diadumene lineata (aka Haliplanella luciae). All tested BK channel blockers inhibited prey killing at concentrations comparable to their inhibition of vertebrate BK channels. In addition, the BK channel blocker paxilline selectively inhibited prey killing mediated by vibration-sensitive Type A cnidocyte supporting cell complexes. We queried a mammalian BK alpha amino acid sequence to the Exaiptasia diaphena database, from which we identified a putative anemone, pore-forming BK alpha subunit sequence. Using the E. diaphena BK alpha sequence as a template, we assembled a BK alpha transcript from our assembled D. lineata transcriptome. In addition, the hydra homolog of D. lineata BK alpha localizes to nematocytes on the hydra single-cell RNA sequencing map. Our findings suggest that D. lineata expresses BK channels that play a role in vibration-sensitive nematocyst discharge from Type A cnidocyte supporting cell complexes. We believe this is the first functional demonstration of BK channels in nonbilaterians. Because stimulated chemoreceptors frequency tune Type A cnidocyte supporting cell complexes to frequencies matching swimming movements of prey via a protein kinase A signaling pathway and protein kinase A generally activates BK channels, we suggest that D. lineata BK channels may participate in protein kinase A-mediated frequency tuning.

Extracellular 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.

Cnidarians require mechanical stimuli to trigger nematocyst discharge and initiate feeding behaviors. The interval from triggering stimulus to response is tens of microseconds, making it likely that mechanically gated ion channels trigger nematocyst discharge. Because many transient receptor potential channels are mechanically gated, we hypothesized that nematocyst discharge involves transient receptor potential channels. We therefore tested various transient receptor potential channel inhibitors to determine whether they inhibit nematocyst discharge and prey killing in the acontiate sea anemone (Actinaria) Diadumene lineata (a.k.a. Haliplanella luciae). Three types of cnidocyte supporting cell complexes regulate nematocyst discharge in anemones: Types C, B, and A. Discharge from Type Cs is directly triggered by stimulation of contact-sensitive mechanoreceptors, while Type Bs require activation of chemoreceptors from prey-derived N-acetylated sugars to sensitize contact-sensitive mechanoreceptors. In Type As, activated chemoreceptors tune vibration-sensitive mechanoreceptors that predispose contact-sensitive mechanoreceptors for triggering. The non-selective transient receptor potential channel blockers lanthanum and gadolinium dose-dependently inhibited about 80% of prey killing and all nematocyst discharge from Type Bs and Type Cs, but not Type As. The selective transient receptor potential vanilloid 4 (TRPV4) blocker GSK2193874 inhibited Type As and Type Bs. However, the selective TRPV4 blockers HC-067047 and RN-1734 inhibited only Type As. Thus, three TRPV4-selective blockers implicate TRPV-like involvement in discharge from Type As, whereas GSK2193874 also affected Type Bs. Our results suggest that a TRPV-like homolog plays an essential role in nematocyst-mediated prey killing from Type As, whereas other transient receptor potential channels are likely involved in discharge from Type B and C cnidocyte supporting cell complexes.

Sensory 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.

Cnidarians, 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.

Becausein vivonematocyst discharge requires extracellular Ca2+, Ca(2+)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 forN-acetylated sugars (e.g.,N-acetylneuraminic acid). Activated chemoreceptors predispose contact-sensitive mechanoreceptors to trigger discharge. We show that activating L-type Ca(2+)channels inhibitsN-acetylneuraminic acid-sensitized discharge, contrary to a previous suggestion. In addition, inhibiting L-type channels increases sensitivity toN-acetylneuraminic acid. Specifically, we show that the L-type Ca(2+)channel activator (-)-Bay K 8644 dose-dependently inhibitsN-acetylneuraminic acid-sensitized discharge, as does raising ambient Ca(2+)levels. We also show that lowering extracellular Ca(2+)levels or adding any of several selective and chemically distinct L-type Ca(2+)channel blockers, including dihydropyridines, dose-dependently increasesN-acetylneuraminic acid sensitivity and broadens the dynamic range ofN-acetylneuraminic acid sensitization. Consistent with these functional findings,Aiptasia pallidaexpresses an L-type Ca(2+)channel alpha subunit transcript that encodes a conserved dihydropyridine-binding site. Phylogenetic analysis confirms a close relationship of theAiptasiaCa(2+)channel alpha subunit sequence between anemones, anthozoans, and cnidarians that extends into protostomal and deuterostomal bilaterians. We conclude that L-type Ca(2+)channel activity modulatesN-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 thatN-acetylneuraminic acid chemoreceptor signaling will activate L-type channels.

Gestational long term hypoxia (LTH) is a common prenatal stress caused by maternal anemia, high altitude living, smoking, and other disorders. Such gestational LTH can lead to a variety of cerebrovascular disorders in the neonate that compromise brain blood flow. Local and whole‐cell Ca 2+ signals are important to the regulation of cerebrovascular tone. Rapid and localized Ca 2+ transients, often referred to as sparks, activate large‐conductance K + channels (BK), which dilate vessels. Whole‐cell Ca 2+ oscillations, in comparison, are important to arterial contraction. Previous work shows that LTH and post‐natal maturity influence BK channel function in basilar arteries, arterial wall Ca 2+ signals, cerebrovascular tone, as well as brain blood flow. We hypothesize that LTH‐dependent changes in spontaneous and depolarization mediated Ca 2+ sparks and whole‐cell oscillations are important to BK channel activity, arterial wall Ca 2+ signals, and vascular reactivity changes our group has previously observed. To begin evaluating the role of individual myocyte Ca 2+ signals to the observed changes in cerebrovascular function, we isolated basilar arteries from adult and near term fetal (~141 gestational days) normoxic and hypoxic sheep that were raised at 3,800 m. for 110+ days. Cytosolic Ca 2+ signals were examined in individual myocytes of intact arterial preparations loaded with fluo‐4 using laser scanning confocal microscopy techniques. Ca 2+ spark activity as well as whole‐cell oscillations were enhanced by depolarizing myocytes with 30 mM K + . Maturation increased Ca 2+ spark activity and depolarization‐dependent oscillations. LTH decreased oscillatory activity in fetal and adult animals but only spark frequency in adult myocytes. LTH decreased the area under the curve for Ca 2+ oscillations independent of age as well as suppressed the ability of adult myocytes to respond to membrane depolarization. Depolarization, maturation, and LTH also influenced the spatial and temporal relationships of the Ca 2+ oscillations. Overall, these observations illustrate that maturation and gestation at high‐altitude modify local and whole‐cell Ca 2+ signaling, which likely contribute to adjustments in BK channel function, arterial wall Ca 2+ signals, vasoreactivity, and cerebral blood flow that our group has examined in the past. Support or Funding Information This material is based upon work supported by NIH grants P01HD083132 (LZ). Imaging was performed in the LLUSM Advanced Imaging and Microscopy Core with support of NSF Grant MRI‐DBI 0923559 and the Loma Linda University School of Medicine. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ca2+ oscillations are important in the regulation of many cerebral arterial functions ranging from vasoconstriction to gene transcription. Without these oscillatory waveforms, there are alterations in the signaling pathways regulated by Ca2+. Previous work has shown that long term hypoxia (LTH) due to high altitude exposure can cause cerebral vascular dysfunction including changes in reactivity and morphology in the fetus as well as adult. Studies performed in cerebral arterial preparations have illustrated that LTH in sheep fetus and adult cause whole cell Ca2+ signaling dysfunctions that compromise vascular reactivity and contribute to cerebral arterial pathologies. Based on the premise that Ca2+ oscillations, vasoconstriction, vessel morphology, and LTH mediated cerebral arterial function are interrelated we tested the hypothesis that LTH impairs Ca2+ oscillations. The impact of LTH and maturation on Ca2+ signals in cerebral arterial myocytes was examined using confocal imaging techniques of flou‐4 loaded myocytes of basilar arteries from low (700m) or high altitude (3,801m) near term fetal or adult sheep. LTH decreased the intracellular Ca2+ signals independent of age due to a faster decay in the Ca2+ signal, an effect that could impair vasoconstriction, alter tissue structure, and compromise the regulation of cerebral blood flow. Along with producing smaller Ca2+ events, the data show that LTH inhibits the cell's ability to respond with Ca2+ signals in response to 30 mM potassium‐induced depolarization in fetal as well as adult sheep. LTH and ontogeny may also play interconnected roles in Ca2+ signaling as LTH inhibits distant communication between myocytes in fetal sheep, whereas in adult sheep it stimulates local signaling. These observations illustrate that altitude and maturation each modify Ca2+ signaling behavior in ways that likely impact arterial reactivity and other mechanisms related to the regulation of cerebral blood flow.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pulmonary vasodilatation at birth underlies the fetal transition to lung breathing. Failure to vasodilate causes persistent pulmonary hypertension of newborn, persistent patency of the ductus arteriosus and foramen ovale , and bronchopulmonary dysplasia‐related hypoxia, often with dire consequences. The mechanism of pulmonary vasodilatation at birth is poorly understood. Pulmonary artery smooth muscle cells (PASMCs) isolated from 4 th and 5 th order resistance vessels of ovine near‐term fetus exhibited resting membrane potentials 8 mV more depolarized than adult (P<0.05), consistent with vasoconstriction. Whole‐cell Kv current densities for voltage‐clamped fetal and adult PASMCs were similar. In contrast, large‐conductance, calcium‐activated K + (BK) channel current densities for fetal cells were significantly lower than adult at +20 to +60 mV, but equal to adult at higher potentials. By Western immunoblots, fetal PASMCs expressed twice the pore‐forming BKα subunit protein as adult, but only half as much accessary BKβ□ subunit. By flow cytometry, cell surface βl expression was also significantly lower in fetal PASMCs. Since optimal BK channel activity occurs at 1:1 β‐to‐α stoichiometry, our results suggested the near‐term fetus expressed BK β:α ratios about ¼ that of adult. These findings suggest near‐term fetal PASMCs are more depolarized than adult due to lower BK activity and lower BKβ□ subunit expression. In agreement with BKα protein levels, BKα mRNA levels were two‐fold higher in near‐term fetus than in adult by qRT‐PCR. However, in contrast to BKβ□ protein levels, BKβ□ mRNA levels were two‐fold higher in near‐term fetus than adult. These findings suggest that BKβ□ mRNA levels may be poised to express higher levels of BKβ□ protein at birth in order to enhance BK channel activity and promote pulmonary vasodilatation upon transition to lung breathing. Support or Funding Information National Institutes of Health Grants HD‐003807‐41 and HD031226‐20 (to LDL)

Ca2+ entry through L‐Type channels mediate contraction of basilar arterial myocytes and whole‐cell Ca2+ waveforms are thought to be integral to this process. Based on previous evidence, we hypothesize that modifications, due to both long term hypoxia (LTH) and ontogeny, will affect the influence of depolarization on intracellular Ca2+ waves. Specifically, fetal arteries will have reduced Ca2+ signals while LTH will further reduce depolarization induced Ca2+ responses. In order to test this hypothesis, we examined Ca2+ wave activity using confocal fluorescence imaging techniques on basilar arterial myocytes in an en face preparation of low and high altitude fetal (FN, FH) and adult (AN, AH) sheep, where sheep were placed at 3,801 m for >100 days. Ca2+ waves were evaluated with and without 30mM K (30K), which was used to depolarize the myocytes. LTH decreased the area under the curve (AUC) independent of animal age. These changes in AUC were due to a faster decay in the Ca2+ signal. Depolarization with 30K increased Ca2+ wave amplitude and duration in all groups except for AH. These observations illustrate that both altitude and ontogeny play important roles in affecting various aspects of the Ca2+ signals. This likely contributes to the changes in arterial contractility that were previously observed with ontogeny and LTH and potentially other mechanisms pertaining to cerebral blood flow.Support or Funding InformationSupport from NIH and NSF

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 Ca2+ 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 Ca2+ 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 β1 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 Ca2+ 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.

Ryanodine receptor (RyR) activity causes Ca2+ sparks in cerebral arterial myocytes by activating adjacent Ca2+‐activated K+ (BK) channels, which can dilate arteries. Previous studies show ontogeny increases Ca2+ sparks and long‐term hypoxia (LTH) increases BK channel activity. We hypothesized that BK channel activity is greater in arterial myocytes of the LTH fetus than adult due to greater spark activity, and because BK channel clustering is greater in regions where sparks occur. To address this hypothesis we examined spark activity and BK channel clustering to sites of potential spark activity. This was accomplished using line‐scan and immunofluorescence techniques, respectively, in basilar arteries from LTH fetal (FH) and adult sheep (AH) kept at 3,801 m for > 100 days. The percentage of myocytes with sparks was ~ 2‐fold greater in FH versus AH and unaffected by 30 mM K (30K). Spark amplitude was 7% greater in FH 30K versus AH 30K, while no spatial or temporal changes to sparks were observed. BK channels were 2‐3 times more clustered and ~1.5 times more co‐localized with cholera toxin B‐labeled clusters in FH. These data suggest greater BK targeting to potential sites of spark activity in FH myocytes. Elevated RyR‐dependent Ca2+ spark activity in FH and altered patterns of BK distribution shed new light on the therapeutic potential of RyRs and BKs for treatment of cerebral vascular disease in newborns and adults.Grant Funding Source: Supported by NSF MRI 0923559, NIH HD‐069746, P01HD031226, R01HD003807, LLUSOM

Both aerobic training and the adipokine, adiponectin (ADPN), increase insulin sensitivity. Because the metabolic effects of aerobic training and ADPN are similar, aerobic training has been proposed to increase ADPN, which has been generally confirmed. However, in separate studies physically active adolescent females showed higher ADPN levels than sedentary controls, but trained young males showed no increase in ADPN levels. Because no direct comparison of males and females have been provided and because physically fit middle-aged adults have been largely neglected from studies on the effects of exercise on ADPN levels, we hypothesized that long-term (6 months), progressive long-distance training would increase ADPN levels more in trained, middle-aged females than in comparably trained, middle-aged males. We recruited aerobically trained, normal-weight, middle-aged females (n=8) and males (n=10) to participate in a prescribed marathon training protocol progressing from 10 to 90 km (6 to 55 miles) per week over 6 mos. We collected and stored fasting plasma samples and recorded body measurements at 0 (baseline) and 6 mos. Stored samples were analyzed for insulin, glucose, and ADPN. ADPN increased significantly among females [from 18.37 ± 10.39 (mean ± SD) to 22.50 ± 9.87 µg/ml; P<0.05], but non-significantly among males (from 6.69 ± 4.47 to 8.10 ± 5.57 µg/ml; P=0.2). No significant reduction in insulin resistance or anthropometric measurements occurred in either group. Our findings suggest that long-term, progressive aerobic training produces greater increases in circulating ADPN levels in trained, middle-aged, normal-weight females than in comparably trained males.

Aerobic training (AT) may increase adiponectin (ADPN) levels, but sex and ethnicity may also affect this interaction. We compared ADPN levels in male (M) and female (F) trained marathoners (T; M=10, F=8) and untrained novices (UT; M=9, F=11) in a 6-mo AT interventional study. Fasting plasma samples were collected at the beginning and end of the 6-mo training and analyzed. ADPN levels increased significantly in both R and B females and B males, but non-significantly in R males. We also compared ADPN levels of Caucasian (C) and non-Caucasian (NC) in both sexes (M, F) from T and UT groups at the beginning of the study. We separated the participants into different groups based on the training history (T, UT), ethnicity (C, NC), and sex (M, F). ADPN levels in the trained female marathoners (C-T-F = 22, NC-T-F = 6) were significantly higher than those in the comparably trained males within same ethnic group (C-T-M = 23, NC-T-M = 11). ADPN levels in the trained female marathoners were also significantly higher than those in the untrained novice females within the same ethnic group (C-UT-F = 18, NC-UT-F = 18). ADPN levels in the trained male marathoners were non-significantly higher than those in the untrained novice males within the same ethnic group (C-UT-M = 9, NC-UT-M = 18). Long-term AT raises ADPN levels in Caucasian and non-Caucasian females, but not males.

Adipose tissue secretes the adipokine, adiponectin (ADPN), which increases insulin sensitivity. Because some of the metabolic effects of exercise and ADPN are similar, exercise has been proposed to increase ADPN. However, most short-term (≤3 mos) and constant-effort exercise protocols have not produced increases in ADPN. Furthermore, no direct comparisons of male and female subjects on the effect of exercise on ADPN levels have been reported. We hypothesized that long-term (6 mos), progressive training would increase ADPN levels in both males and females. We recruited middle-aged, untrained males and females to participate in an interventional study employing a marathon training regimen progressing from 9.7 to 88.5 km (6 to 55 miles) per week over 6 mos. At baseline, we matched the mean ages of the male and female groups. We collected and stored fasting plasma samples and recorded body measurements at 0 (baseline) and 6 mos. Stored samples were analysed for insulin, glucose, and ADPN. ADPN increased significantly among both males (from 5.89 ± 2.46 (mean ± SD) to 7.65 ± 3.18 μg/ml; p < 0.05) and females (from 8.48 ± 3.22 to 10.56 ± 4.05 μg/ml; p < 0.05). The extent of the increase in ADPN was similar in the male (40.7 ± 50%; median, 12.1%) and female (27.0 ± 31.1%; median, 22.3%) groups. However, there was no significant reduction in insulin resistance as measured by the HOMA-IR scores in either group. We conclude that long-term, progressive aerobic training increases circulating ADPN levels in middle-aged, untrained males and females.

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.