EFFECTS OF TEMPERATURE ON CHRONIC HYPOXIA TOLERANCE IN THE NON-INDIGENOUS BROWN MUSSEL, PERNA PERNA (BIVALVIA: MYTILIDAE) FROM THE TEXAS GULF OF MEXICO

Effects of temperature (15°, 20° and 25°C), O₂ partialpressure (P_O2=0, 1, 2, 4, and 6 kPa), and individual size(12–79 mm shell length; SL) on survivorship of specimensof the non-indigenous, marine, brown mussel, Perna perna, fromTexas were investigated to assess its potential distributionin North America. Its hypoxia tolerance was temperature-dependent,survivorship being significantly extended at lower temperaturesunder all tested lethal P_O2. Incipient tolerated P_O2 was 4 and6 kPa at 15 and 20°C, respectively, with >50% mortalityoccurring at 25°C at all tested levels of hypoxia. P_O2 hadless of an effect on survival of hypoxia than temperature. At25°C, survivorship was not different over a P_O2 range of0–2 kPa and increased only at 4 and 6 kPa. Survivorshipwas size-dependent. Median survival times increased with increasingSL in anoxia and P_O2=1 kPa, but at 2, 4 and 6 kPa,smaller individuals survived longer than larger individuals.With tolerance levels similar to other estuarine bivalve species,P. perna should withstand hypoxia encountered in estuarine environments.Thus, its restriction to intertidal rocky shores may be dueto other parameters, particularly its relatively low temperaturetolerance. (Received 26 January 2004; accepted 31 March 2005)     

Cl- currents activated via purinergic receptors in Xenopus follicles

Ionic currents elicited via purinergic receptors located in themembrane of Xenopus follicles werestudied using electrophysiological techniques. Follicles responded toATP-activating inward currents with a fast time course(F_in). InRinger solution, reversal potential (E_rev) ofF_in was 22mV, which did not change with external substitutions ofNa⁺ orK⁺, whereas solutions containing50 or 5% of normal Clconcentration shiftedE_rev to about +4and +60 mV, respectively, and decreasedF_in amplitude,indicating thatF_in was carriedby Cl.F_in had an onsetdelay of ~400 ms, measured by application of a brief jet of ATP froma micropipette positioned near the follicle (50 µm).F_in was inhibitedby 50% in follicles pretreated with pertussis toxin. This suggests a Gprotein-mediated receptor channel pathway.F_in was mimickedby 2-MeSATP and UTP, the potency order (half-maximal effectiveconcentration) was 2-MeSATP (194 nM) > UTP (454 nM) > ATP(1,086 nM). All agonists generatedCl currents and displayedcross-inhibition on the others.F_in activation byacetylcholine also cross-inhibitedF_in-ATPresponses, suggesting that all act on a common channel-activationpathway.

     

ATP and PIP2 dependence of the magnesium-inhibited, TRPM7-like cation channel in cardiac myocytes

The Mg²⁺-inhibited cation (MIC) current (I_MIC) in cardiac myocytes biophysically resembles currents of heterologously expressed transient receptor potential (TRP) channels, particularly TRPM6 and TRPM7, known to be important in Mg²⁺ homeostasis. To understand the regulation of MIC channels in cardiac cells, we used the whole cell voltage-clamp technique to investigate the role of intracellular ATP in pig, rat, and guinea pig isolated ventricular myocytes. I_MIC, studied in the presence or absence of extracellular divalent cations, was sustained for 50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells I_MIC exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5'-(,-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate prevented I_MIC rundown. In contrast, under similar conditions neither the inhibition of protein phosphatases 1, 2A, 2B or of protein tyrosine phosphatase nor the activation of protein kinase A (forskolin, 20 µM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) by prevention of its resynthesis (10 µM wortmannin or 15 µM phenylarsine oxide) induced rundown of I_MIC. Finally, loading ATP-depleted cells with exogenous PIP₂ (10 µM) prevented rundown. These results suggest that PIP₂, likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity. cation channels; hydrolysis; phosphoinositides; rundown     

cAMP production in rabbit carotid body: role of adenosine

Chen, J., B. Dinger, and S. J. Fidone. cAMP productionin rabbit carotid body: role of adenosine. J. Appl.Physiol. 82(6): 1771-1775, 1997.In the presentstudy, we have investigated the possible role of adenosine in thehypoxia-mediated increase in adenosine 3,5-cyclicmonophosphate (cAMP) in the carotid body. cAMP levels in rabbit carotidbodies superfused in vitro for 10 min were increased in the presence ofadenosine (100 µM and 1.0 mM; maximum increase = 127%,P < 0.01). These effects werereduced by the nonspecific adenosine-receptor antagonist 1,3-dipropyl-8[p-sulfophenyl]xanthine(DPSPX; 10 µM). The specific A₂-receptor agonist2-[4(2-carboxymethyl)phenylethylamino]-5-N-ethylcarboxamido adenosine (CGS-21680; 100 nM) also elevated carotid body cAMP levels,an effect that was blocked by the specificA₂-antagonist 3,7-dimethyl-L-propargyl-xanthine(DMPX; 50 µM). Hypoxia-evoked elevations in cAMP were potentiated inthe presence of the adenosine-uptake inhibitor dipyridamole (100 nM)and blocked by exposure to adenosine-receptor antagonists. Our datasuggest that the rabbit carotid body contains specific adenosinereceptors (A₂ subtype) that arepositively coupled to adenylate cyclase and that increases in cAMPassociated with hypoxia are mediated by the release of endogenousadenosine.

     

Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury

Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca²⁺ accumulation resulting from functional coupling of Na⁺/Ca²⁺ exchange (NCE) with stimulated Na⁺/H⁺ exchange (NHE1) and 2) 17-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na⁺ and therefore Ca²⁺ accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pH_i), Na⁺ (Na), and calcium concentration ([Ca²⁺]_i) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pH_i, Na and [Ca²⁺]_i during ischemia (P < 0.05). In control OVX vs. OVX+E2, pH_i fell from 6.93 ± 0.03 to 5.98 ± 0.04 vs. 6.96 ± 0.04 to 6.68 ± 0.07; Na rose from 25 ± 6 to 109 ± 14 meq/kg dry wt vs. 25 ± 1 to 76 ± 3; [Ca²⁺]_i changed from 365 ± 69 to 1,248 ± 180 nM vs. 293 ± 66 to 202 ± 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 µM N-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca²⁺]_i is greater than can be accounted for by the thermodynamic effect of reduced Na accumulation on NCE. myocardial ischemia; Na⁺/H⁺ exchange; Na⁺/Ca²⁺ exchange; nuclear magnetic resonance; ischemic biology; ion channels/membrane transport; transplantation     

Gamete Release at Low Tide in Fucoid Algae: Maladaptive or Advantageous?

This review discusses three questions pertaining to gamete releaseby fucoid algae at low tide: 1) Are gametes viable and doesfertilization occur at low tide?, 2) How many gametes are releasedat low tide versus at high tide? and 3) Is gamete release atlow tide maladaptive or is it selectively advantageous? Gameterelease at low tide (LT) in fucoid algae is observed commonlyin monoecious species from the lower and mid-intertidal zones(e.g., Fucus distichus, F. evanescens, F. gardneri, Pelvetiacompressa); in dioecious species, intact antheridia (containingsperm) are commonly released at low tide (e.g., in Ascophyllumnodosum, F. vesiculosus). Fertilization at low tide can be determinedwith a calcofluor white assay and occurs in at least three species(F. distichus, F. gardneri, P. compressa). In general, fucoidalgae have high levels of fertilization success, but substantialmortality occurs during early embryogenesis in some intertidalzones due to physical stresses. The agarose bead assay is usefulto assess desiccation on an egg-sized scale. Constitutive dehydrin-likeproteins are present in sperm, eggs, and embryos. The releaseof gametes at low tide appears to be a consequence of a mechanismselected to permit gamete release under calm conditions in seawater;this mechanism is described. The timing of adhesion by zygotesat low and high tide is unknown. Such information and additionaldata on the relative proportions of zygotes produced by moneciousspecies at low tide versus high tide are required in order toassess effects of gamete release at low tide upon dispersaland population structure.     

beta -Adrenergic regulation of constitutive nitric oxide synthase in cardiac myocytes

Nitric oxide (NO) has been implicated in endogenous control ofmyocardial contractility. However, NO release has not yet been demonstrated in cardiac myocytes. Accordingly, endogenous NO production was measured with a porphyrinic microsensor positioned on the surfaceof individual neonatal or adult rat ventricular myocytes (n > 6 neonatal and adult cells perexperiment). In beating neonatal myocytes, there was no detectablespontaneous NO release with each contraction. However, norepinephrine(NE; 0.25-1 µM) elicited transient NO release from beatingneonatal (149 ± 11 to 767 ± 83 nM NO) and noncontracting adult(157 ± 13 to 791 ± 89 nM NO) cells. NO was released byadrenergic agonists with the following rank order of potency:isoproterenol(₁₂) > NE (/₁) > dobutamine (₁)  epinephrine(/₁₂) > tertbutylene (₂); NO wasnot released by phenylephrine (). NE-evoked NO release wasreversibly blocked byN^G-monomethyl-L-arginine,trifluoperazine, guanosine5'-O-(2-thiodiphosphate), andnifedipine but was enhanced by 3-isobutyl-1-methylxanthine (0.5 mM = 14.5 ± 1.6%) and BAY K 8644 (10 µM = 11.9 ± 1%). NO wasalso released by A-23187 (10 µM = 884 ± 88 nM NO), guanosine 5'-O-(3-thiotriphosphate) (1 µM = 334 ± 56 nMNO), and dibutyryl adenosine 3',5'-cyclic monophosphate(10-100 µM = 35 ± 9 to 284 ± 49 nM NO) but not by ATP,bradykinin, carbachol, 8-bromoguanosine 3',5'-cyclicmonophosphate, or shear stress. This first functional demonstration ofa constitutive NO synthase in cardiac myocytes suggests its regulationby a -adrenergic signaling pathway and may provide a novel mechanismfor the coronary artery vasodilatation and enhanced diastolicrelaxation observed with adrenergic stimulation.

     

Control of the respiratory cycle in conscious humans

Rafferty, G. F., and W. N. Gardner. Control of therespiratory cycle in conscious humans. J. Appl.Physiol. 81(4): 1744-1753, 1996.We studied inconscious humans the relative strength of mechanisms controlling timingand drive components of the respiratory cycle around their resting setpoints. A system of auditory feedback with end-tidalPCO₂ held constant in mild hyperoxiavia an open circuit was used to induce subjects independently to change inspiratory time (TI) andtidal volume(VT_I)over a wide range above and below the resting values for every breathfor up to 1 h. Four protocols were studied in various levels ofhypercapnia (1-5% inspiredCO₂). We found thatTI (and expiratory time) could be changed over a wide range (1.17-2.86 s,P < 0.01 forTI) and VT_Iincreased by 500 ml (P < 0.01)without difficulty. However, in no protocol was it possible to decreaseVT_I belowthe free-breathing resting value in response to reduction of auditoryfeedback thresholds by up to 600 ml. This applied at all levels ofchemical drive studied, with restingVT_I valuesvarying from 1.06 to 1.74 liters. When reduction inVT_I wasforced by the more "programmed" procedure of isocapnic panting,end-expiratory volume was sacrificed to ensure that peak tidal volumereached a fixed absolute lung volume. These results suggest that theimperative for control of resting breathing is to prevent reduction ofVT_I belowthe level dictated by the prevailing chemical drive, presumably tosustain metabolic requirements of the body, whereas respiratory timingis weakly controlled consistent with the needs for speech and othernonmetabolic functions of breathing.

     

Potent block of inactivation-deficient Na+ channels by n-3 polyunsaturated fatty acids

A voltage-gated, small, persistent Na⁺ current (I_Na) has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent I_Na that may cause arrhythmias. In the present study, we investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on I_Na in HEK-293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the -subunit (hH1) of human cardiac Na⁺ channels (hNav1.5) plus ₁-subunits. Extracellular application of 5 µM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited I_Na. The late portion of I_Na (I_{Na late}, measured near the end of each pulse) was almost completely suppressed. I_Na returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on I_Na was concentration dependent, with IC₅₀ values of 4.0 ± 0.4 µM for I_Na peak (I_{Na peak}) and 0.9 ± 0.1 µM for I_{Na late}. EPA shifted the steady-state inactivation of I_{Na peak} by –19 mV in the hyperpolarizing direction. EPA accelerated the process of resting inactivation of the mutant channel and delayed the recovery of the mutated Na⁺ channel from resting inactivation. Other polyunsaturated fatty acids, docosahexaenoic acid, linolenic acid, arachidonic acid, and linoleic acid, all at 5 µM concentration, also significantly inhibited I_Na. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids stearic acid and palmitic acid at 5 µM concentration had no effect on I_Na. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1 induce inactivation-deficient I_Na and that n-3 PUFAs inhibit mutant I_Na. human cardiac sodium channel     

Artificial Insemination 'Regulated by EDTA' in the Monoecious Brown Alga Fucus evanescens

As part of an attempt to control the fertilization of a monoeciousbrown alga Fucusevanescens, the effects of EDTA on the releaseand fertilization of gametes were studied. When receptacleswere treated for liberation of gametes by soaking in plain seawater,egg-packets and spermatophores were discharged from oogoniaand antheridia, respectively, with the gametes retained withintheir packing envelopes. After a while, the envelopes were disruptedand the gametes were released. Thirty min after the soaking,over 99% of eggs were fertilized. However, when receptacleswere soaked in seawater that contained 0.5 mg m1^–1 Na₂.EDTAat 4°C, the release and fertilization of gametes were preventedafter egg-packets and spermatophores had been released fromthe receptacles. Release of gametes from such egg-packets andspermatophores occurred rapidly when the medium was dilutedwith an excess of plain seawater. The chelating agent affectedthe disruption of egg-packets and spermatophores, but it didnot affect the subsequent fertilization and development of thefertilized eggs. On the basis of these results, normal unfertilizedeggs and sperm were isolated separately by filtration and centrifugationof the released packing envelopes in the presence of 0.5 mgml^–1 Na₂EDTA at 4°C. Artificial insemination usingthe isolated gametes was successful. ³ Present address: Akan Board of Education, Akan, 085-02 Japan.     

Differential Response of Pure Lines of Avena fatua L. to Substituted Phthalimides: Germination and Endosperm-mobilization Studies

The germination and endosperm-mobilization responses of twogenetically pure lines (AN-51 and Mont 73) of Avena fatua (wildoat) to gibberellic acid (GA₃, ) and three substituted phthalimides(experimental compounds AC-92, 803, AC-94, 377 and AC-99, 524)were studied. The line AN-51 showed a much greater responseto GA₃, in terms of the percentage and the rate of stimulationof germination, than Mont 73. These lines also differed significantlyin their response to AC-94, 377 and AC-99, 524. The relativegermination response of the two lines to these phthalimideswas very similar to that for GA₃. The phthalimide AC-94, 377was more effective at stimulating germination than AC-99, 524,whereas AC-92, 803 had little or no effect. Salicylhydroxamate,an inhibitor of alternative respiration, did not inhibit theAC-94, 377-induced germination. Like GA₃, AC-94, 377 induced -amylase production and the releaseof reducing sugars by 3 mm endosperm segments. GA₃, was mosteffective at inducing endosperm mobilization, followed by AC-94,377, AC-99, 524 and AC-92, 803, respectively. The line AN-51showed a significantly greater response to both AC-94, 377 andGA₃, than Mont 73. It is concluded that: (1) genetically pure lines of wild oatsdiffer in their response to substituted phthalimides - the lineAN-51 being more responsive than Mont 73, (2) the phthalimideAC-94, 377 is the most effective at inducing germination andAC-92, 803 the least, (3) like GA₃, phthalimides induce endospermmobilization and pure lines differ in the degree of this response,and (4) salicylhydroxamate-sensitive respiration is not necessaryfor the stimulation of germination by AC-94-77. The similaritiesin the effects of AC-94, 377 and GA₂, (two structurally dissimilarcompounds), and in the relative response of the two lines tothese chemicals may provide a useful system for the investigationof wild oat seed physiology. The differential susceptibilityof pure lines to phthalimides also indicates that use of thesecompounds to deplete wild oat seed banks may increase the proportionof less responsive biotype(s) in field populations. Wild oats, weed seed bank, dormancy, germination, endosperm mobilization, -amylase, Avena fatua, phthalimides, gibberellic acid     

Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity

Weinvestigated a role of neuroregulation in the release of eosinophilchemotactic activity (ECA) from bovine bronchial epithelial cells(BBEC). BBEC were stimulated with acetylcholine (ACh) and substance P(SP), and the supernatant fluids were tested for ECA by a blind-wellchemotactic chamber technique. BBEC released ECA in response to ACh andSP in a dose- and time-dependent manner. Checkerboard analysis showedthat ECA in regard to ACh and SP was chemotactic rather thanchemokinetic. Partial characterization revealed that ECA involved bothlipids and peptides. The release of ECA in response to ACh and SP wasinhibited by nonspecific and 5-specific lipoxygenase inhibitors and bycycloheximide (P < 0.01). Molecular-sieve columnchromatography revealed that these mediators induced three molecularmass peaks (near 25 kDa, 9 kDa, and 400 Da, respectively). The lowestpeak, which represented the predominant activity, was blocked byleukotriene B₄-receptor antagonist (P < 0.01) but not by platelet-activating factor-receptor antagonist. The releaseof leukotriene B₄ in the supernatant fluids was increased in response to ACh and SP stimulation (P < 0.01).Platelet-activating factor was not detected. These results raise thepossibility of a role of neuroregulation for the elaboration of ECA inthe airway.

     

P2X receptors in mouse Leydig cells

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 µM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K_d) of 44, 110, and 637 µM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP ATPS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). -Methylene-ATP (-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK_a) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)_Ca/P_Na = 5.32], but not to chloride (P_Cl/P_Na = 0.03) or N-methyl-D-glucamine (NMDG) (P_NMDG/P_Na = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between –90 and –50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant () = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X₂. patch clamp; single channels; ATP; desensitization     

Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia

Activation ofprotein kinase C with phorbol 12-myristate 13-acetate (PMA) causedcomplex transient perturbations of amiloride-sensitive short-circuitNa⁺ currents(I_Na) in A6epithelia and frog skins that were tissue and concentration dependent.A noninvasive channel blocker pulse method of noise analysis (18) wasused to investigate how PMA caused time-dependent changes of apicalmembrane epithelial Na⁺ channel(ENaC) single-channel currents, channel open probabilities (P_o), andchannel densities(N_T). In A6epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependentsustained decreases ofP_o (~55% belowcontrol, 50 nM) and rapid compensatory transient increases ofN_T within 7 min(~220% above control, 50 nM), resulting in either small transientincreases of I_Naat 5 nM PMA or small biphasic decreases ofI_Na at 50 nM PMA.In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin causedafter a delay of at least 10 min transient increases ofN_T to~60-70% above control at 30-60 min. Unlike A6 epithelia,P_o was increased~15% above control within 7 min and remained within±10-15% of control for the duration of the 2-h experiments.Despite differences in the time courses of secondary inhibition oftransport in A6 epithelia and frog skin, the delayed downregulation oftransport was due to time-dependent decreases ofN_T from theirpreelevated levels in both tissues. WhereasP_o is decreasedwithin minutes in A6 epithelia as measured by noise analysis or bypatch clamp (8), the discrepancy in regulation ofN_T in A6epithelia as measured by noise analysis and patch clamp is most likelyexplained by the inability of on-cell patches formed before treatmentof tissues with PMA to respond to regulation of their channeldensities.

     

Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms

Published data suggest that the neuropeptide calcitonin gene-related peptide (CGRP) can stimulate osteoblastic bone formation; however, interest has focused on activation of cAMP-dependent signaling pathways in osteogenic cells without full consideration of the importance of cAMP-independent signaling. We have now examined the effects of CGRP on intracellular Ca²⁺ concentration ([Ca²⁺]_int) and membrane potential (E_m) in preosteoblastic human MG-63 cells by single-cell fluorescent confocal analysis using fluo 4-AM-fura red-AM and bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC₄(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca²⁺]_int: a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC₅₀ of 0.30 nM, and the maximal effect (initially 3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca²⁺ mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca²⁺ influx with verapamil or inhibiting cAMP-dependent signaling with the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS). These data suggest that CGRP initially stimulates Ca²⁺ discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular E_m, with maximal effect at 20 nM and an EC₅₀ of 0.30 nM. This effect was attenuated with charybdotoxin (–20%) or glyburide (glibenclamide; –80%), suggesting that E_m hyperpolarization is induced by both Ca²⁺-activated and ATP-sensitive K⁺ channels. Thus CGRP signals strongly by both cAMP-dependent and cAMP-independent signaling pathways in preosteoblastic human MG-63 cells. osteoblastic cells; calcium; membrane potential; potassium channels; adenosine 3',5'-cyclic monophosphate     

The signaling role of extracellular ATP and its dependence on Ca2+ flux in elicitation of Salvia miltiorrhiza hairy root cultures

The application of a polysaccharide elicitor from yeast extract,YE, to Salvia miltiorrhiza hairy root cultures induced transientrelease of ATP from the roots to the medium, leading to a dose-dependentincrease in the extracellular ATP (eATP) level. The eATP levelrose to a peak (about 6.5 nM with 100 mg l^–1 YE) at about10 h after YE treatment, but dropped to the control level 6h later. The elicitor-induced ATP release was dependent on membraneCa²⁺ influx, and abolished by the Ca²⁺ chelator EGTA or thechannel blocker La³⁺. The YE-induced H₂O₂ production was stronglyinhibited by reactive blue (RB), a specific inhibitor of membranepurinoceptors. On the other hand, the application of exogenousATP at 10–100 µM to the cultures also induced rapidand dose-dependent increases in H₂O₂ production and medium pH,both of which were effectively blocked by RB and EGTA. The non-hydrolyzableATP analog ATPS was as effective as ATP, but the hydrolyzedderivatives ADP or AMP were not so effective in inducing thepH and H₂O₂ increases. Our results suggest that ATP releaseis an early event and that eATP plays a signaling role in theelicitation of plant cell responses; Ca²⁺ is required for activationof the elicitor-induced ATP release and the eATP signal transduction.This is the first report on ATP release induced by a fungalelicitor and its involvement in the elicitor-induced responsesin plant cells.     

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels

Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa⁺ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na⁺ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa⁺ current (I_Na) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited I_Na by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced I_Na inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced I_Na inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited I_Na; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na⁺channels by PKC isozyme.

     

Chloride secretion by semicircular canal duct epithelium is stimulated via beta 2-adrenergic receptors

The ductalepithelium of the semicircular canal forms much of the boundary betweenthe K⁺-rich luminal fluid and the Na⁺-richabluminal fluid. We sought to determine whether the net ion fluxproducing the apical-to-basal short-circuit current(I_sc) in primary cultures was due to anionsecretion and/or cation absorption and under control of receptoragonists. Net fluxes of ²²Na, ⁸⁶Rb, and³⁶Cl demonstrated a basal-to-apical Clsecretion that was stimulated by isoproterenol. Isoproterenol andnorepinephrine increased I_sc with anEC₅₀ of 3 and 15 nM, respectively, and isoproterenolincreased tissue cAMP of native canals with an EC₅₀ of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had noeffect on I_sc. Isoproterenol stimulation ofI_sc and cAMP was inhibited by ICI-118551(IC₅₀ = 6 µM for I_sc) but notby CGP-20712A (1 µM) in primary cultures, and similar results werefound in native epithelium. I_sc was partially inhibited by basolateral Ba²⁺ (IC₅₀ = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, andDIDS did not fully fit the profile for CFTR. Our findings show that thecanal epithelium contributes to endolymph homeostasis by secretion ofCl under ₂-adrenergic control with cAMP assecond messenger, a process that parallels the adrenergic control ofK⁺ secretion by vestibular dark cells. The current workpoints to one possible etiology of endolymphatic hydrops in Meniere'sdisease and may provide a basis for intervention.

     

A3 adenosine receptors regulate Cl- channels of nonpigmented ciliary epithelial cells

Adenosinestimulates Cl channels ofthe nonpigmented (NPE) cells of the ciliary epithelium. We sought toidentify the specific adenosine receptors mediating this action.Cl channel activity inimmortalized human (HCE) NPE cells was determined by monitoring cellvolume in isotonic suspensions with the cationic ionophore gramicidinpresent. The A₃-selective agonistN⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide(IB-MECA) triggered shrinkage (apparentK_d = 55 ± 10 nM). A₃-selective antagonists blocked IB-MECA-triggered shrinkage, andA₃-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 µMadenosine when all four known receptor subtypes are occupied. TheA₁-selective agonistN⁶-cyclopentyladenosineexerted a small effect at 100 nM but not at higher or lowerconcentrations. The A_2A agonistCGS-21680 triggered shrinkage only at high concentration (3 µM), aneffect blocked by MRS-1191. IB-MECA increased intracellularCa²⁺ in HCE cells and alsostimulated short-circuit current across rabbit ciliary epithelium.A₃ message was detected in bothHCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possessA₃ receptors and that adenosinecan activate Cl channels inNPE cells by stimulating these A₃ receptors.     

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K_i between 0.2 and 0.5 µM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 µM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 µM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability. respiration; voltage-dependent anion-selective channel; apoptosis; cell death