Cross talk between polysulfide and nitric oxide in rat peritoneal mast cells.

The aim of this study was to define the effects of polysulfide on intracellular Ca(2+) concentration ([Ca(2+)]i) and the underlying machinery, especially from the hydrogen sulfide (H2S) and nitric oxide (NO) perspectives, in rat peritoneal mast cells. We found that a polysulfide donor, Na2S4, increased [Ca(2+)]i, which is both extracellular and intracellular Ca(2+) dependent. Intracellular Ca(2+) release induced by Na2S4 was attenuated by the addition of a ryanodine receptor blocker. A slow-releasing H2S donor, GYY4137, dose dependently increased [Ca(2+)]i that was independent from extracellular Ca(2+) influx. The GYY4137-induced [Ca(2+)]i release was partially attenuated in the presence of the ryanodine receptor blocker. Both polysulfide and H2S donors increased the intracellular NO levels in DAF-2-loaded mast cells, which were abolished by an NO scavenger, cPTIO. Inhibition of NO synthase (NOS) significantly abolished the polysulfide- or H2S-donor-induced [Ca(2+)]i elevation in the absence of extracellular Ca(2+) An NO donor, diethylamine (DEA) NONOate, increased [Ca(2+)]i in a concentration-dependent manner, in which both extracellular and intracellular Ca(2+) are associated. At higher concentrations, the DEA NONOate-induced [Ca(2+)]i increases were attenuated in the absence of extracellular Ca(2+) and by the addition of the ryanodine receptor blocker. H2S and NO dose dependently induced polysulfide production. Curiously, polysulfide, H2S, and NO donors had no effect on mast cell degranulation. Among synthases, cystathionine-γ-lyase, and neuronal NOS seemed to be the major H2S- and NO-producing synthases, respectively. These results indicate that polysulfide acts as a potential signaling molecule that regulates [Ca(2+)]i homeostasis in rat peritoneal mast cells via a cross talk with NO and H2S.

Reciprocal interaction among gasotransmitters in isolated pancreatic ß-cells.

We aimed to elucidate the interplay among the three well-known gas molecules, nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S), and their effects on intracellular Ca(2+) concentration ([Ca(2+)]i) and insulin secretion in rat pancreatic ß-cells. Immunofluorescence studies demonstrated the expression of constitutive enzymes that are responsible for the production of NO, CO and H2S. CO and H2S increased NO production as indicated by the increase in diaminofluorescein-2 triazole fluorescence. NO and CO induced an elevation in the sulfane sulfur pool and concomitantly H2S production. The NO- and CO-induced H2S production was partially inhibited by hypotaurine, an H2S scavenger. NO and H2S produced CO production as revealed by a myoglobin assay. A calmodulin antagonist in the absence of extracellular Ca(2+) significantly attenuated NO and H2S production. NO and CO induced a [Ca(2+)]i increase mainly via Ca(2+) release from internal stores; however, H2S induced a [Ca(2+)]i increase via the influx of extracellular Ca(2+). NO dose-dependently stimulated basal insulin release but CO dose-dependently inhibited it. H2S showed an insignificant effect on basal insulin secretion from freshly isolated pancreatic islets. Herein, we address for the first time the reciprocal and synergistic relation among gasotransmitters with diverse effects on basal insulin secretion that regulate ß-cells functions and homeostasis.

Female Mice Avoid Male Odor from the Same Strain via the Vomeronasal System in an Estrogen-Dependent Manner.

Inbreeding avoidance is essential to providing offspring with genetic diversity. Females' mate choice is more crucial than males' for successful reproduction because of the high cost of producing gametes and limited chances to mate. However, the mechanism of female inbreeding avoidance is still unclear. To elucidate the mechanism underlying inbreeding avoidance by females, we conducted Y-maze behavioral assays using BALB/c and C57BL/6 female mice. In both strains, the avoidance of male urine from the same strain was lower in the low estrogen phase than in the high estrogen phase. The estrous cycle-dependent avoidance was completely prevented by vomeronasal organ (VNO) removal. To assess the regulation of the vomeronasal system by estrogen, the neural excitability was evaluated by immunohistochemistry of the immediate early gene products. Although estrogen did not affect neural excitability in the VNO, estrogen enhanced the neural excitability of the mitral cell layer in the AOB induced by urine from the cognate males. These results suggest that female mice avoid odor from genetically similar males in an estrogen-dependent manner via the vomeronasal system and the excitability of the mitral cells in the AOB is presumed to be regulated by estrogen.

Hydrogen sulfide: a novel gaseous signaling molecule and intracellular Ca2+ regulator in rat parotid acinar cells.

In addition to nitric oxide (NO), hydrogen sulfide (H2S) is recognized as a crucial gaseous messenger that exerts many biological actions in various tissues. An attempt was made to assess the roles and underlying mechanisms of both gases in isolated rat parotid acinar cells. Ductal cells and some acinar cells were found to express NO and H2S synthases. Cevimeline, a muscarinic receptor agonist upregulated endothelial NO synthase in parotid tissue. NO and H2S donors increased the intracellular Ca(2+) concentration ([Ca(2+)]i). This was not affected by inhibitors of phospholipase C and inositol 1,4,5-trisphosphate receptors, but was decreased by blockers of ryanodine receptors (RyRs), soluble guanylyl cyclase, and protein kinase G. The H2S donor evoked NO production, which was decreased by blockade of NO synthases or phosphoinositide 3-kinase or by hypotaurine, an H2S scavenger. The H2S donor-induced [Ca(2+)]i increase was diminished by a NO scavenger or the NO synthases blocker. These results suggest that NO and H2S play important roles in regulating [Ca(2+)]i via soluble guanylyl cyclase-cGMP-protein kinase G-RyRs, but not via inositol 1,4,5-trisphosphate receptors. The effect of H2S may be partially through NO produced via phosphoinositide 3-kinase-Akt-endothelial NO synthase. It was concluded that both gases regulate [Ca(2+)]i in a synergistic way, mainly via RyRs in rat parotid acinar cells.

A novel role for carbon monoxide as a potent regulator of intracellular Ca2+ and nitric oxide in rat pancreatic acinar cells.

Carbon monoxide (CO) is known as an essential gaseous messenger that regulates a wide array of physiological and pathological processes, similar to nitric oxide (NO) and hydrogen sulfide. The aim of the present study was to elucidate the potential role of CO in Ca(2+) homeostasis and to explore the underlying mechanisms in pancreatic acinar cells. The exogenous application of a CO-releasing molecule dose-dependently increased intracellular Ca(2+) concentration ([Ca(2+)]i). A heme oxygenase (HO) inducer increased [Ca(2+)]i in a concentration-dependent manner, and the increase was diminished by an HO inhibitor. The CO-induced [Ca(2+)]i increase persisted in the absence of extracellular Ca(2+), indicating that Ca(2+) release is the initial source for the increase. The inhibition of G protein, phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP3) receptor diminished the CO-induced [Ca(2+)]i increase. CO upregulated endothelial nitric oxide synthase (eNOS) expression and stimulated NO production, and NOS inhibitor, calmodulin inhibitor, or the absence of extracellular Ca(2+) eliminated the latter response. Blocking the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway abolished CO-induced NO production. Pretreatment with an NOS inhibitor, NO scavenger, or soluble guanylate cyclase inhibitor, did not affect the CO-induced [Ca(2+)]i increase, indicating that NO, soluble guanylate cyclase, and cyclic guanosine 5'-monophosphate are not involved in the CO-induced [Ca(2+)]i increase. CO inhibited the secretory responses to CCK-octapeptide or carbachol. We conclude that CO acts as a regulator not only for [Ca(2+)]i homeostasis via a PLC-IP3-IP3 receptor cascade but also for NO production via the calmodulin and PI3K-Akt/PKB pathway, and both CO and NO interact. Moreover, CO may provide potential therapy to ameliorate acute pancreatitis by inhibiting amylase secretion.

Hydrogen sulfide regulates Ca(2+) homeostasis mediated by concomitantly produced nitric oxide via a novel synergistic pathway in exocrine pancreas.

AIM: The present study was designed to explore the effects of hydrogen sulfide (H2S) on Ca(2+) homeostasis in rat pancreatic acini. RESULTS: Sodium hydrosulfide (NaHS; an H2S donor) induced a biphasic increase in the intracellular Ca(2+) concentration ([Ca(2+)]i) in a dose-dependent manner. The NaHS-induced [Ca(2+)]i elevation persisted with an EC50 of 73.3 µM in the absence of extracellular Ca(2+) but was abolished by thapsigargin, indicating that both Ca(2+) entry and Ca(2+) release contributed to the increase. The [Ca(2+)]i increase was markedly inhibited in the presence of NG-monomethyl L-arginine or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), and diaminofluorescein-2/diaminofluorescein-2 triazole (DAF-2/DAF-2T) fluorometry demonstrated that nitric oxide (NO) was also produced by H2S in a dose-dependent manner with an EC50 of 64.8 µM, indicating that NO was involved in the H2S effect. The H2S-induced [Ca(2+)]i increase was inhibited by pretreatment with U73122, xestospongin C, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, KT5823, and GP2A, indicating that phospholipase C (PLC), the inositol 1,4,5-trisphosphate (IP3) receptor, soluble guanylate cyclase (sGC), protein kinase G (PKG), and Gq-protein play roles as intermediate components in the H2S-triggered intracellular signaling. INNOVATION: To our knowledge, our study is the first one highlighting the effect of H2S on intracellular Ca(2+) dynamics in pancreatic acinar cells. Moreover, a novel cascade was presumed to function via the synergistic interaction between H2S and NO. CONCLUSION: We conclude that H2S affects [Ca(2+)]i homeostasis that is mediated by H2S-evoked NO production via an endothelial nitric oxide synthase (eNOS)-NO-sGC-cyclic guanosine monophosphate-PKG-Gq-protein-PLC-IP3 pathway to induce Ca(2+) release, and this pathway is identical to the one we recently proposed for a sole effect of NO and the two gaseous molecules synergistically function to regulate Ca(2+) homeostasis.

Preputial gland activates olfactory receptor neurons in rat: calcium imaging study using laser scanning confocal microscopy.

The rodent preputial gland is one of the major sources of odours and is reported to be involved in several behavioural activities. However, how the preputial gland initiates the olfactory response to manifest the effects is not known. Olfactory receptor neurons (ORNs) present in the olfactory epithelium are involved in the perception of odorant/pheromonal compounds. In the present study, the response of rat ORNs to preputial gland extract was evaluated by calcium imaging analysis. We found that some rat ORNs responded to the preputial gland extract by exhibiting an intracellular calcium response. By contrast, the ORNs did not respond at all to the foot pad extract (control). The results indicated that the substances contained in the preputial gland might interact with a type of receptor expressed in the female rat ORNs, suggested to manifest the behavioural responses, such as social and sexual interactions. This study provided the first evidence of activation of ORNs by the preputial gland extract.

Estrus cycle-related preference of BALB/c female mice for C57BL/6 males is induced by estrogen.

The odor preference of female mice for male odor is reported to have cyclical variations in relation to the estrus cycle. Females prefer the odor of genetically dissimilar males to that of genetically similar ones, but the causal relation between this preference and the estrus cycle has scarcely been investigated. The Y-maze test demonstrated that BALB/c females stayed for a longer duration near the urine of C57BL/6 males than that of BALB/c males when they were in metestrus, diestrus and proestrus, but not in estrus. The prolonged stay disappeared after ovariectomy, and administration of estradiol-17ß restored the tendency. The present results suggest that the odor preference of BALB/c females for C57BL/6 over BALB/c males temporally changes according to the estrus cycle and that estrogen can be one of endogenous factors regulating this phenomenon.

Increased squalene concentrations in the clitoral gland during the estrous cycle in rats: an estrus-indicating scent mark?

Squalene in the rat clitoral gland is reported to be semi-volatile and may serve as a chemo-signal. The objective was to determine squalene concentrations in the clitoral gland throughout the reproductive cycle. Clitoral glands were extracted with dichloromethane; 23 compounds were identified with Gas Chromatography linked Mass Spectrometry (GC-MS). Since squalene concentrations were significantly higher during proestrus and estrus, and remarkably reduced during metestrus and diestrus, we inferred that it could be an ovulation-indicating chemosignal in the female rat, acting as a scent mark for the male. This hypothesis was tested by investigating its efficacy to attract males, including studying the role of the olfactory-vomeronasal system of the male in perceiving squalene. For detection of squalene, males used their conventional olfactory system when at a distance from the female, whereas the vomeronasal organ was used when they were in close proximity to the female. We concluded that squalene was a female-specific chemosignal that attracted males, and furthermore, that the olfactory-vomeronasal system had an important role in the perception of squalene.

Nitric oxide stimulates IP3 production via a cGMP/ PKG-dependent pathway in rat pancreatic acinar cells.

In an attempt to explore the functioning of nitric oxide (NO) in pancreatic exocrine cells, we have recently obtained several lines of circumstantial evidence indicating that one of molecular targets of NO is phospholipase C (PLC), the activation of which leads to an increase in the cytosolic Ca2+ concentration ([Ca2+]i) via inositol 1, 4, 5-trisphosphate, IP3. However, whether IP3 is actually produced by NO has not yet been substantiated. The present study was therefore designed to directly measure the intracellular IP3, concentration ([IP3]i) for better understanding of the underlying mechanisms with the help of pharmacological tools. [IP3]i was measured using a fluorescence polarization technique (HitHunter). We obtained the following results: 1) varying concentrations of an NO donor, sodium nitroprusside (SNP), elevated [IP3]i, 2) this elevation was completely inhibited in the presence of the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (ODQ), 3) varying concentrations of the cGMP analogue, 8-Br-cGMP, also increased [IP3]i, 4) the cGMP analogue-induced IP3 production was abolished by pretreatment with either a PLC inhibitor, U73122, or a G-protein inhibitor, GP2A, and 5) KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase G (PKG), also abolished the IP3 production induced by 8-Br-cGMP. These results suggest that the NO-induced [Ca2+]i increase is triggered by an increase in [IP3]i located downstream from intracellular cGMP elevation. In this intracellular pathway, each sGC, cGMP-dependent PKG, G-protein and PLC were suggested to be involved. The present work provides new insights into the intracellular signaling accelerated by NO. NO triggers a [Ca2+]I increase via cGMP and IP3 in pancreatic acinar cells.

A fundamental role for NO-PLC signaling pathway in mediating intracellular Ca2+ oscillation in pancreatic acini.

The aim of the present study was to investigate the possible interaction between intracellular Ca(2+) and nitric oxide (NO) in rat pancreatic acinar cells, especially intracellular signaling events. (1) Nitric oxide donors SNP (0.1-100 µM) and NOR-3 (50-400 µM) induced Ca(2+) oscillations in fluo-4-loaded acini, that appeared to be analogous to what we usually observe in acini stimulated with physiological secretagogues such as CCK-8 and this oscillations were abolished in the presence of carboxy-PTIO. (2) The NO donors-evoked Ca(2+) oscillations were not abolished even in the absence of extracellular Ca(2+) but totally disappeared when cells were pretreated with thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitor. (3) Inhibition of guanylate cyclase with 1 H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) attenuated Ca(2+) oscillations evoked by SNP in the absence of extracellular Ca(2+). (4) Inhibitors of phospholipase C activity, U73122 and the IP(3)R blocker xestospongin C, both abolished the SNP-induced Ca(2+) response. (5) Furthermore, we found that both CCK-8 and carbachol (CCh) induced NO production in DAF-2-loaded acinar cells and that an inhibitor of NO synthase, N(G)-monomethyl-l-arginine (L-NMMA), significantly reduced CCK-8-induced Ca(2+) oscillation. These results indicate that NO mobilizes Ca(2+) from internal stores through activation of guanylate cyclase and resultant cGMP production. In addition, PLC activation of IP(3) production is also suggested to be involved in Ca(2+) mobilization via IP(3) receptors. This suggests the presence of cross-talk between Ca(2+) and NO in pancreatic acini and this cascade may, at least partially, participate in physiological secretagogue-evoked Ca(2+) dynamics in pancreatic acinar cells.

Identification of testosterone-dependent volatile compounds and proteins in the preputial gland of rat Rattus norvegicus.

Preputial gland is one of the best known and most odour-producing organs in many non-primate mammals. It is generally believed that the development of this gland and functions are regulated by testosterone. To substantiate this point, the present study was aimed to evaluate the testosterone-dependent volatile compounds and proteins in the preputial gland of rat adopting castration and testosterone supplementation. The results revealed that four compounds, geranyl linalool isomer, oxirane, farnesol and lanosterol, are testosterone-dependent. Similarly, a low molecular mass protein with molecular weight 18kDa, supposed to be a pheromone carrier, also is shown to be testosterone-dependent. This study leads to the conclusion that testosterone-dependent compounds and sex-associated protein are present in the preputial gland of rat which may act as a sex pheromone and pheromone carrier, respectively.

Metabolic alkalosis due to feeding chicks in breeding Adélie penguins Pygoscelis adeliae under natural conditions.

Prolonged abnormal vomiting causes metabolic alkalosis. Many seabirds are known to feed their chicks by regurgitation. We hypothesized that metabolic alkalosis occurs in seabirds even under natural conditions during the breeding season. Adélie penguins Pygoscelis adeliae feed their chicks by regurgitating food for 50-60 d until the chicks fledge. In this study, the concentrations of Cl(-), HCO(3)(-), Na+, K+, pH, and PCO2 in the blood of breeding Adélie penguins were measured throughout the chick-rearing season. The pH of penguin venous blood shifted from 7.54 in the guarding period to 7.47 in the crèche period. Decreasing Cl(-) and increasing HCO(3)(-) blood concentrations in parents were associated with increasing mass of their brood in the guarding period, the early phase of the rearing season, indicating that regurgitating to feed chicks causes loss of gastric acid and results in relative metabolic alkalosis. The inverse trend was observed during the crèche period, the latter phase of the rearing season, when parents spent more time at sea and have fewer opportunities for gastric acid loss. This was assumed to be the recovery phase. These results indicate that regurgitation might cause metabolic alkalosis in breeding Adélie penguins. To our knowledge, this is the first report to indicate that seabirds exhibit metabolic alkalosis due to regurgitation to feed chicks under natural conditions.

Morphological features and blood parameters of Weddell seal (Leptonychotes weddellii) mothers and pups during the breeding season.

We investigated the blood status of 9 Weddell seal mothers and 9 pups during the breeding season in a field study conducted from November to December 2004 at a breeding colony in Antarctica. The blood glucose and total cholesterol concentrations were higher in the pups than in the mothers. On the other hand, the blood urea nitrogen concentration was lower in the pups than in the mothers. Growth-associated depletion of blood triglyceride was observed in the pups and may have been due to the post-weaning fast. The results characterize the blood status of Weddell seals in relation to physiological adaptations for breeding.

A dual system of intercellular calcium signaling in glial nets associated with lanceolate sensory endings in rat vibrissae.

The lanceolate sensory endings that form palisades around the hair follicle associate with networks of branched Schwann cells. To define the properties of these glial networks as possible conduits of Ca2+ signals, lanceolate endings isolated from rat vibrissae were observed by confocal microscopy while the signaling was locally activated by mechanical stimulation. Intercellular coupling by gap junctions was also assessed by a technique employing fluorescence recovery after photobleaching (FRAP) and by transmission electron microscopy (TEM). Results showed that the glial Ca2+ signals can spread among the arrays of lanceolates in two forms: rapid signals that originate in individual Schwann processes covering the lanceolate axon terminals around the locus of mechanical stimulation, and delayed ones that travel from the stimulation locus through cytoplasmic arborization of the primarily activated cell to the adjacent cell processes. The former signaling was suppressed by the antipurinergic agents suramin and apyrase, whereas the latter was sensitive to the gap junction blocker carbenoxolon. FRAP experiments and TEM observations corroborated the presence of gap junction communications between the Schwann processes of different cell origins. These findings show that, in the Schwann networks, purinergically induced Ca2+ signals and those dependent on gap junctions are propagated in their own spatiotemporal patterns to constitute two distinct forms of communication among the mechanoreceptor palisades.

Voltage-gated channel properties of epithelial cells in porcine vomeronasal organ.

Bipolar vomeronasal sensory neurons (VSNs) in the vomeronasal organ (VNO) are believed to detect pheromones in most mammals. The vomeronasal sensory epithelium (VSE) is composed of VSNs and supporting cells. There are morphological differences in VNOs between species. Many electrophysiological experiments have been performed on rodent VSEs but few on other mammals. We therefore investigated voltage-gated channel properties of cells in the porcine VSE using slice whole-cell voltage-clamp techniques. In immunohistochemical study of the porcine VSE, most PGP9.5-immunoreactive cells were found between the middle and basal region, and negative cells were distributed in the apical to middle region. Depolarizing pulses to epithelial cells from -90mV produced transient inward Na+ channel currents and sustained outward K+ channel currents with various amplitudes. The distribution of cells having high and low Na+ current densities was mostly consistent with the histological distribution of VSNs and supporting cells, respectively. The half-inactivation voltage of voltage-gated Na+ channels in supporting cells was 26mV more negative than that in VSNs. Voltage-gated K+ channel currents in both cell types were suppressed by tetraethylammonium to the same extent. VSNs possessed TTX-sensitive voltage-gated Na+ channels and Ni2+ -sensitive T-type Ca2+ channels. These results suggest that the histological distribution of porcine vomeronasal epithelial cells is more similar to the dog and goat than to rodents, and that the electrophysiological characteristics of porcine vomeronasal epithelial cells are similar to those of rodents. It is also suggested that porcine VSNs detecting pheromones generate action potentials through these channels.

T-type Ca2+ channels contribute to IBMX/forskolin- and K(+)-induced Ca(2+) transients in porcine olfactory receptor neurons.

T-type Ca(2+) channels are low-voltage-activated Ca(2+) channels that control Ca(2+) entry in excitable cells during small depolarization above resting potentials. Using Ca(2+) imaging with a laser scanning confocal microscope we investigated the involvement of T-type Ca(2+) channels in IBMX/forskolin- and sparingly elevated extracellular K(+)-induced Ca(2+) transients in freshly isolated porcine olfactory receptor neurons (ORNs). In the presence of mibefradil (10microM) or Ni(2+) (100microM), the selective T-type Ca(2+) channel inhibitors, IBMX/forskolin-induced Ca(2+) transients in the soma were either strongly (>60%) inhibited or abolished completely. However, the Ca(2+) transients in the knob were only partially (<60%) inhibited. Ca(2+) transients induced by 30mM K(+) were also partially ( approximately 60%) inhibited at both the knob and soma. Furthermore, ORNs responded to as little as a 2.5mM increase in the extracellular K(+) concentration (7.5mM K(+)), and such responses were completely inhibited by mibefradil or Ni(2+). These results reveal functional expression of T-type Ca(2+) channels in porcine ORNs, and suggest a role for these channels in the spread Ca(2+) transients from the knob to the soma during activation of the cAMP cascade following odorant binding to G-protein-coupled receptors on the cilia/knob of ORNs.

Intensity of odorant stimulation affects mode of Ca2+ dynamics in rat olfactory receptor neurons.

We investigated the relation between the intensity of odorant stimulation and the mode of spatiotemporal Ca(2+) dynamics in Fluo-4-loaded rat olfactory receptor neurons (ORNs) using a confocal laser scanning microscope. We found that relatively smaller Ca(2+) transients remained confined to the knob while larger ones spread to the soma with latency. Prolonged odor exposure ensured the spread of Ca(2+) transients from the knob to the soma. Upon exposing ORNs to progressively increasing concentrations of odor, the Ca(2+) transients that were confined to the knob at lower concentrations extended to the soma at higher concentrations. Stimulation with progressively increasing concentrations of forskolin plus IBMX yielded identical results. Partial inhibition of adenylyl cyclase by MDL12330A changed the odor response extending to the soma to a response confined to the knob. Blocking of L-type Ca(2+) channels by nifedipine reduced the magnitude of the response extending to the soma but had no effect on the response confined to the knob. It is thus suggested that Ca(2+) transients confined to the knob represent weak stimulation, and, speculatively, such responses either constitute inhibitory responses or indicate weak excitatory responses that fail to outstand the spontaneous electrical noise of ORNs.

Emperor penguins adjust swim speed according to the above-water height of ice holes through which they exit.

Emperor penguins leap from the water onto the sea ice. Their ability to reach above-water height depends critically on initial vertical speed of their leaping, assuming that the kinetic energy is converted to gravitational potential energy. We deliberately changed the above-water heights of ice hole exits, in order to examine whether penguins adjusted swim speed in accordance with the above-water height of the ice. Penguins were maintained in a corral on the fast ice in Antarctica, and voluntarily dived through two artificial ice holes. Data loggers were deployed on the penguins to monitor under water behavior. Nine instrumented penguins performed 386 leaps from the holes during experiments. The maximum swim speeds within 1 s before the exits through the holes correlated significantly with the above-water height of the holes. Penguins adopted higher speed to exit through the higher holes than through the lower holes. Speeds of some failed exits were lower than the theoretical minimum values to reach a given height. Penguins failed to exit onto the sea ice in a total of 37 of the trials. There was no preference to use lower holes after they failed to exit through the higher holes. Rather, swim speed was increased for subsequent attempts after failed leaps. These data demonstrated that penguins apparently recognized the above-water height of holes and adopted speeds greater than the minimal vertical speeds to reach the exit height. It is likely, especially in the case of higher holes (>40 cm), that they chose minimum speeds to exit through the holes to avoid excess energy for swimming before leaping. However, some exceptionally high speeds were recorded when they directly exited onto the ice from lower depths. In those cases, birds could increase swim speed without strokes for the final seconds before exit and they only increased the steepness of their body angles as they surfaced, which indicates that the speed required for leaps by emperor penguins were aided by buoyancy, and that penguins can sometimes exit through the ice holes without any stroking effort before leaping.

Characterization of forskolin-induced Ca2+ signals in rat olfactory receptor neurons.

Forskolin-induced Ca(2+) signals were examined in isolated rat olfactory receptor neurons (ORNs) using a Ca(2+) indicator, fura-2. In the soma of the ORNs, forskolin caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that was enhanced by a phosphodiesterase (PDE) 1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methyl-xanthine, but not a PDE4 inhibitor, rolipram. Forskolin-induced Ca(2+) signals were abolished with the removal of extracellular Ca(2+) and un-affected by treatment with thapsigargin or caffeine plus ryanodine. Niflumic acid, a Ca(2+)-activated Cl(-) channel inhibitor, or nifedipine, an L-type Ca(2+) channel inhibitor, slowed the initial rate of the increase in [Ca(2+)](i) in response to forskolin. Nifedipine did not affect the increase in [Ca(2+)](i) that was slowed by niflumic acid. In Ca(2+) measurements with a confocal microscope and a calcium indicator, Fluo-4, the onset of the response to forskolin in the knob region occurred simultaneously or earlier, but not later, than that in the soma. It is suggested that the forskolin-induced Ca(2+) signals are due to Ca(2+) influx, but not the release of Ca(2+) from Ca(2+) stores, and that the initial rapid increase in [Ca(2+)](i) is associated with the activation of the voltage-dependent Ca(2+) channels in rat ORNs.