Participation of cAMP in a signal-transduction pathway relating erythrocyte deformation to ATP release

Previously, we reportedthat red blood cells (RBCs) of rabbits and humans release ATP inresponse to mechanical deformation and that this release of ATPrequires the activity of the cystic fibrosis transmembrane conductanceregulator (CFTR). It was reported that cAMP, acting through acAMP-dependent protein kinase, PKA, is an activator of CFTR. Here weinvestigate the hypothesis that cAMP stimulates ATP release from RBCs.Incubation of human and rabbit RBCs with the direct activator ofadenylyl cyclase, forskolin (10 or 100 µM), with IBMX (100 µM),resulted in ATP release and increases in intracellular cAMP. Inaddition, epinephrine (1 µM), a receptor-mediated activator ofadenylyl cyclase, stimulated ATP release from rabbit RBCs. Moreover,incubation of human and rabbit RBCs with an active cAMP analog[adenosine 3'5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMP, 100 µM)] resulted in ATP release. In contrast, forskolin and Sp-cAMPwere without effect on dog RBCs, cells known not to release ATP inresponse to deformation. When rabbit RBCs were incubated with theinactive cAMP analog and inhibitor of PKA activity, adenosine3',5'-cyclic monophosphorothioate Rp-isomer (100 µM),deformation-induced ATP release was attenuated. These results areconsistent with the hypothesis that adenylyl cyclase and cAMP arecomponents of a signal-transduction pathway relating RBC deformation toATP release from human and rabbit RBCs.

     

Studies on the inhibition a fatty acid synthesis in the chicken liver by adenine compoundsin vitro

The biosynthesis of fatty acids from [l-¹⁴C]-acetate in the chicken liver slicesin vitro was inhibited by cAMP, adenosine, 5′-AMP, 3′-AMP, ATP, NAD and FAD but not by adenine, guanine or inosine. The minimum structural requirement for inhibition appears to be adenosine. The inhibitory action of adenosine, 5′-AMP and NAD on fatty acid synthesis is likely to be mediated by adenosine or its metabolites since adenosine deaminase reverses the inhibition while it has no effect on the inhibition by cAMP; thus, the inhibitory effect of cAMP is probably not mediated through its hydrolysis products, 5′-AMP, or adenosine.     

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by β-adrenergic signals, leading to a rise in intracellular 3′,5′-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 ± 0.08. A change in assay temperature from a euthermic value (37 °C) to one typical of hibernating body temperature (5 °C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 °C to 8.7 at 5 °C, (b) K_m values at 37 °C for Mg.ATP (49.2±3.4 M) and for two phosphate acceptors, Kemptide (50.0±5.5 M) and Histone IIA (0.41 ± 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 °C, and (c) inhibition by KCl, NaCl and NH₄Cl was reduced. However, temperature change had little or no effect on K_m values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 °C and activation energies that were 29.1 ± 1.0 kJ/mol for temperatures >9 °C and 2.3-fold higher at 68.1 ± 2.1 kJ/mol for temperatures <9 °C, whereas the rabbit enzyme showed a breakpoint at 17 °C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 ± 0.18 at 37 °C to 2.19 ± 0.07 at 5 °C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with ³²P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 °C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor. Accepted: 10 July 1998     

Apical Heterotrimeric G-proteins Activate CFTR in the Native Sweat Duct

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl⁻ conductance (CFTR G _Cl ) in the native sweat duct (SD). We permeabilized the basolateral membrane with α-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G _Cl activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-γ-S (100 μm) also activates CFTR G _Cl in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-γ-S increased CFTR G _Cl by 44 ± 20 mS/cm² (mean ±se; n= 7). GDP (10 mm) inhibited G-protein activation of CFTR G _Cl even in the presence of GTP-γ-S. The heterotrimeric G-protein activator (AlF₄ ⁻) in the cytoplasmic bath activated CFTR G _Cl (increased by 51.5 ± 9.4 mS/cm² in the presence of 5 mm ATP without cAMP, n= 6), the magnitude of which was similar to that induced by GTP-γ-S. Employing immunocytochemical-labeling techniques, we localized Gαs, Gαi, Gαq, and Gβ at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G _Cl in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G _Cl activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G _Cl activity. Received: 9 June 2000/Revised: 5 October 2000     

Myocardial energy metabolism in ischemic preconditioning and cardioplegia: A metabolic control analysis

For both, cardioplegia (CP) and ischemic preconditioning (IP), increased ischemic tolerance with reduction in infarct size is well documented. These cardioprotective effects are related to a limitation of high energy phosphate (HEP) depletion. As CP and IP have to be assumed to act by different mechanisms, their effects on myocardial HEP metabolism cannot be assumed to be identical. Therefore, a systematic analysis of myocardial HEP metabolism for both procedures and their combination was performed, addressing the question whether there are different effects on myocardial HEP metabolism by IP and CP. In this study, metabolic control analysis was used to analyze the regulation of HEP metabolism. In open chest pigs subjected to 45 min LAD occlusion (index ischemia), CP and IP preserved myocardial ATP (control (C) 0.14 ± 0.05 μmol/g wwt; CP: 0.95 ± 0.14, IP: 0.61 ± 0.12; p<0.05 C vs. CP and IP) and reduced myocardial necrosis (infarct size IA/RA: C: 90.0 ± 3.0%; CP: 0.0 ± 0.0% but patchy necroses; IP: 5.05 ± 2.1%; p<0.05 C vs. CP and IP). The effects on HEP metabolism, however, were different: CP acted predominantly by slowing down the breakdown of phosphocreatine (PCr) during early phases of ischemia (C: ΔPCr 0–2 min: 5.24 ± 0.32 μmol/g wwt; CP: ΔPCr 0–2 min: 3.38 ± 0.23 μmol/g wwt, p<0.05 vs. C), leaving ATP breakdown during later stages unaffected (C: ΔATP 5–45 min: 1.77 ± 0.11 μmol/g wwt CP: ΔATP 5–45 min: 1.59 ± 0.28 μmol/g wwt, n.s. vs. C). In contrast to CP, in IP PCr breakdown was even increased (IP: ΔPCr 0–2 min: 7.06 ± 0.34 μmol/g wwt, p<0.05 vs. C), but ATP depletion greatly attenuated (IP: ΔATP 5–45 min: 0.48 ± 0.10 μmol/g wwt, p<0.05 vs. C and CP). Combining IP and CP yielded an additive effect with slowing down the breakdown of both PCr (IP+CP: ΔPCr 0–2 min: 5.09± 0.35 μmol/g wwt, p<0.05 vs. C and IP) and ATP (IP+CP: ΔATP 5–45 min: 0.56 ± 0.48 μmol/g wwt, p<0.05 vs. C and CP), resulting in a higher ATP content at the end of index ischemia (1.86 ± 0.46 μmol/g wwt, p<0.05 vs. C, CP and IP). Compared to IP, combining IP+CP achieved also a further reduction in infarct size (IA/RA: 0.0 ± 0.0%, p<0.05 vs IP) and—compared to CP—a disappearance of the patchy necroses. {The concept of major differences in myocardial HEP metabolism during CP and IP is further supported at a molecular level by metabolic control analysis. CP but not IP slowed down the CK reaction velocity at high PCr levels. In contrast to CP exerting a continuous decline in vATPase for any given ATP level, in IP myocardium ATPase reaction velocity was even increased at higher ATP contents, whereas a marked decrease in ATPase reaction velocity was found if ATP levels decreased. The equilibrium of the CK-reaction remained unchanged following CP, whereas IP induced a changing CK equilibrium, which was the more shifted towards PCr the more myocardial HEP content decreased. The data demonstrate different effects of CP and IP on myocardial HEP metabolism, i.e. PCr and ATP breakdown as well as the apparent equilibrium of the creatine kinase (CK)-reaction. For these reasons the combination of the two protective interventions has an additive effect. (Mol Cell Biochem 278: 222–232, 2005)     

Normal CFTR Activity and Reversed Skin Potentials in Pseudohypoaldosteronism

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl⁻ channel function is required for activating amiloride-sensitive epithelial Na⁺ channels (ENaC) in salt-absorbing human sweat duct. It is unclear whether ENaC channel function is also required for CFTR activation. The dysfunctional ENaC mutations in type-1 pseudohypoaldosteronism (PHA-1) provided a good opportunity to study this phenomenon of ion channel interaction between CFTR and ENaC. The PHA-1 ducts completely lacked spontaneous ENaC conductance (gENaC). In contrast, the normal ducts showed large spontaneous gENaC (46 ± 10 ms, mean ± SE). After permeabilization of the basolateral membrane with α-toxin, cAMP + ATP activation of CFTR Cl⁻ conductance (gCFTR) or alkalinization of cytosolic pH (6.8 to 8.5) stimulated gENaC of normal but not PHA-1 ducts. In contrast, both spontaneous gCFTR in intact ducts and (cAMP + ATP)-activated gCFTR of permeabilized ducts appeared to be similar in normal and PHA-1 subjects. Lack of gENaC completely blocked salt absorption and caused dramatic reversal of skin potentials associated with pilocarpine-induced sweat secretion from significantly negative in normal subjects (−13 ± 7.0 mV) to significantly positive (+22 ± 11.0 mV) in PHA-1 patients. We conclude that virtual lack of ENaC in PHA-1 ducts had little effect on CFTR activity and that the positive skin potentials could potentially serve as a diagnostic tool to identify type-1 pseudohypoaldosteronism. An erratum to this article is available at .     

cAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells

The eye’s aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl^– enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl^– channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl^– channels with a unitary conductance of 272 ± 2 pS (n = 80). Voltage steps from 0 to ±80 mV, but not to ±40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl^– channels. cAMP also activated the maxi-Cl^– channels in outside-out patches. In both cases, maxi-Cl^– channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl^– concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl^– concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at ±80 but not at ±40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl^– channels of native PE cells that contribute to Cl^– release particularly from Cl^–-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl^– reabsorption into the ciliary stroma. cell volume; chloride secretion; aqueous humor formation     

Adenylate cyclase and phosphodiesterase activities in relation to a rapid increase in cellular cAMP concentration by hypotonic shock indunaliella viridis

An adenylate cyclase activity of 16.02±1.03 pmol cAMP produced min⁻¹ (mg protein)⁻¹ was detected in a cell homogenate ofDunaliella viridis, a unicellular halotolerant green alga. It was present in both the membrane fraction and soluble fraction separated from the homogenate. Adenylate cyclase activity in the homogenate was activated by 1μM GTPγS but not by Ca²⁺+calmodulin, suggesting this enzyme to be regulated by a G-protein. A phosphodiesterase activity of 23.12±15.03 pmol cAMP decomposed min⁻¹ (mg protein)⁻¹ was found in the homogenate. These activities suggest the presence of a cAMP mediated signal transduction system inDunaliella. Cells, transferred from 1.7 M NaCl medium to 1 M NaCl, showed rapid increase in cAMP within 2 min to about 1.5 times the original concentration (from 2.4±0.2 to 3.9±0.2 pmol per 10⁸ cells) which was recovered in 30 min.     

Function of cAMP as a Mat-Forming Factor in the Cyanobacterium Spirulina platensis

A dense suspension of Spirulina platensis trichomes aggregatedrapidly and formed a diskshaped algal mat when cAMP was added.Cyclic AMP significantly stimulated algal mat formation at concentrationsas low as 10^–7 M. Stimulation of the mat formation wasmost rapid at about 10^–5M cAMP, but higher concentrationswere not increasingly effective. Other nucleotides such as cGMP,ATP and AMP showed no stimulatory effect on algal mat formation.CCCP, an inhibitor of ATP synthesis, was found to suppress thecAMP-stimulated algal mat formation. Cyclic AMP also stimulatedrespiration and gliding movement of this cyanobacterium. (Received September 2, 1991; Accepted October 15, 1991)     

Ion currents induced by ATP and angiotensin II in cultured follicular cells of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors, but little is known about those endogenously expressed in their enveloping layer of follicular cells (FCs). Whole-cell recordings and the perforated patch-clamp technique in cultured FCs held at −60 mV revealed that ATP (20–250 μM) generates inward currents of 465 ± 93 pA (mean ± standard error) in ∼60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in ∼5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at −60 mV disclosed that ATP elicited biphasic inward currents, corresponding to the well-known F_in and S_in-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other hand, when using wholecell recordings, 1 μM Ang II yielded smooth inward currents of 157 ± 45 pA in ∼16% of the FC held at −60 mV. The net Ang II response, mediated by the activation of the AT₁ receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and Ang II receptors in the physiology of X. laevis oocytes.     

Retinoylation Reactions are Inversely Related to the Cardiolipin Level in Testes Mitochondria from Hypothyroid Rats

The effect of hypothyroidism, induced by 6-n-propyl-2-thiouracil (PTU) administration to rats, on the retinoylation reaction and oxidative status was investigated in rat-testes mitochondria. In hypothyroid mitochondria, when compared to euthyroid controls, we found a noticeable increase in the amount of all-trans-retinoic acid (atRA) bound to mitochondrial proteins by an acylation process (34.2 ± 1.9 pmoles atRA/mg protein/360 min and 22.2 ± 1.7 pmoles atRA/mg protein/360 min, respectively). This increase, which was time- and temperature-dependent, was accompanied by a strong reduction in the cardiolipin (CL) amount in the mitochondrial membranes of hypothyroid (2.6 ± 0.2%) as compared to euthyroid rats (4.5 ± 0.5%) Conversely, a decreased retinoylation reaction was observed when CL liposomes were added to mitochondria or mitoplasts from both euthyroid and hypothyroid rats, thus confirming a role of CL in the retinoylation process. In mitochondria from the latter animals an increase of the level of oxidized CL occurred. The ATP level, which was reduced in hypothyroid mitochondria (27.3 ± 4.1 pmoles ATP/mg protein versus 67.1 ± 8.3 pmoles ATP/mg protein of euthyroid animals), was surprisingly increased in mitochondria by the retinoylation reaction in the presence of 100 nM atRA (481.5 ± 19.3 pmoles ATP/mg protein of hypothyroid animals versus 84.7 ± 7.7 pmoles ATP/mg protein of euthyroid animals). Overall, in hypothyroid rat-testes mitochondria the increase in retinoylation activity correlates with a significant depletion of the CL level, due to a peroxidation of this lipid. In addition, an enhanced production of reactive oxygen species was observed.     

Direct measurement of energy fluxes from mitochondria into cytoplasm in permeabilized cardiac cells <Emphasis Type="Italic">in situ:</Emphasis> some evidence for mitochondrial interactosome

The aim of this study was to measure energy fluxes from mitochondria in isolated permeabilized cardiomyocytes. Respiration of permeabilized cardiomyocytes and mitochondrial membrane potential were measured in presence of MgATP, pyruvate kinase – phosphoenolpyruvate and creatine. ATP and phosphocreatine concentrations in medium surrounding cardiomyocytes were determined. While ATP concentration did not change in time, mitochondria effectively produced phosphocreatine (PCr) with PCr/O₂ ratio equal to 5.68 ± 0.14. Addition of heterodimeric tubulin to isolated mitochondria was found to increase apparent Km for exogenous ADP from 11 ± 2 μM to 330 ± 47 μM, but creatine again decreased it to 23 ± 6 μM. These results show directly that under physiological conditions the major energy carrier from mitochondria into cytoplasm is PCr, produced by mitochondrial creatine kinase (MtCK), which functional coupling to adenine nucleotide translocase is enhanced by selective limitation of permeability of mitochondrial outer membrane within supercomplex ATP Synthasome-MtCK-VDAC-tubulin, Mitochondrial Interactosome.     

MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca²⁺-dependent MLC kinase or Ca²⁺-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 µM) increased ERK2 phosphorylation from basal 17 ± 3 to 53 ± 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 µM) or U0126 (10 µM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca²⁺ rise, because it was unaltered when this was suppressed by the Ca²⁺ chelator BAPTA (10 µM) or xestospongin C (3 µM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 µM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 µM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca²⁺-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase. mitogen-activated protein kinase; contractile machinery; myosin light chain kinase; myosin light chain phosphatase     

Regulation of exocytosis by purinergic receptors in pancreatic duct epithelial cells

In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca²⁺]_i. Low concentrations of ATP (<10 µM) induced intracellular Ca²⁺ oscillation but no significant exocytosis. In contrast, 100 µM ATP induced a sustained [Ca²⁺]_i rise and increased the exocytosis rate sevenfold. The contribution of Ca²⁺ or cAMP pathways to exocytosis was tested by using the Ca²⁺ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca²⁺]_i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 µM stimulates exocytosis from PDEC through both Ca²⁺ and cAMP pathways. secretion; amperometry; photometry; calcium, adenosine 3',5'-cyclic monophosphate     

Evidence for Multidrug Resistance-1 P-Glycoprotein-dependent Regulation of Cellular ATP Permeability

The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)¹ family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release ∼3-fold. The MDR1 inhibitors cyclosporine A (10 μm) and verapramil (10 μm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl⁻ current density from −33.1 ± 12.5 pA/pF to −2.0 ± 0.3 pA/pF (−80 mV, p≤ 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd³⁺ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways. Received: 20 November 2000/Revised: 25 May 2001     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage

Little is known of the functional properties of the mammalian,brain-specific Na⁺/H⁺ exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, andits activity was characterized using the fluorescent pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na⁺ (apparent K_Na = 27 ± 5 mM) and a Hill coefficient near 3 for the intracellularproton concentration with a half-maximal activity at an intracellularpH of 6.93 ± 0.03. NHE5 activity was inhibited by acute exposureto 8-bromo-cAMP or forskolin (which increases intracellular cAMP byactivating adenylate cyclase). The kinase inhibitor H-89 reversed thisinhibition, suggesting that regulation by cAMP involves a proteinkinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did nothave a significant effect on activity. The protein kinase C (PKC)activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKCantagonist chelerythrine chloride blunted this effect. Activity wasalso inhibited by hyperosmotic-induced cell shrinkage but wasunaffected by a hyposmotic challenge. These results demonstrate thatrat brain NHE5 is downregulated by activation of PKA and PKC and bycell shrinkage, important regulators of neuronal cell function.

     

Modeling the Current-Voltage Characteristics of Chara Membranes: I. The Effect of ATP Removal and Zero Turgor

We have obtained and modeled the electrical characteristics of the plasma membrane of Chara internodal cells: intact, without turgor and perfused with and without ATP. The cells were voltage and space-clamped to obtain the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membrane. The intact cells yielded similar I/V characteristics with resting p.d.s of −221 ± 12 mV (cytoplasmic clamp, 5 cells) and −217 ± 12 mV (vacuolar clamp, 5 cells). The cut unperfused cells were depolarized at −169 ± 12 mV (7 cells) compared to the vacuole-clamped intact cells. The cells perfused with ATP fell into three groups: hyperpolarized group with resting p.d. −175 ± 12 mV (4 cells) and I/V profile similar to the intact and cut unperfused cells; depolarized group with resting p.d. of −107 ± 12 mV (6 cells) and I/V profiles close to linear; and excited cells with profiles showing a negative conductance region and resting p.d. at −59 ± 12 mV (5 cells). The cells perfused with medium containing no ATP showed upwardly concave I/V characteristics and resting p.d. at −81 ± 12 mV (6 cells). The I/V curves were modeled employing the ``Two-state' model for the H<sup>+</sup> pump (Hansen et al., 1981). The inward and outward rectifiers were fitted to exponential functions and combined with a linear background current. The excitation state in perfused cells was modeled by including an inward current, i <sub>excit</sub>, with p.d.-dependence described by a combination of hyperbolic tangent functions. An inward current, i <sub>no-ATP</sub>, with a smaller amplitude, but very similar p.d.-dependence was also included in the simulation of the I/V curves from cells without ATP. This approach avoided I/V curve subtraction. The modeling of the total I/V and G/V characteristics provided more information about the parameters of the ``Two-state' pump model, as well as more quantitative understanding of the interaction of the major transport systems in the plasmalemma in generation of the resting potential under a range of circumstances. ATP had little effect on nonpump currents except the excitation current; depolarization profoundly affected the pump characteristics. Received: 23 January/Revised: 10 October 1995     

An immortal cell line to study the role of endogenous cftr in electrolyte absorption

Summary The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of “endogenous” CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed two fundamental components of absorption, a cAMP activated Cl⁻ conductance (G_cl) and an amiloride-sensitive Na⁺ conductance (G_Na). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10⁻⁵ M) significantly increased the Cl⁻ diffusion potential (V_t) generated by a luminally directed Cl⁻ gradient from − 15.3 ± 0.7 mV to −23.9 ± 1.1 mV,n=39; and decreased the transepithelial resistance (R_t) from 814.8 ± 56.3 Ω.cm² to 750.5 ± 47.5 Ω.cm²,n=39, (n=number of cultures). cAMP activation, anion selectivity (Cl⁻>I⁻>gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited G_Cl), all indicate that the G_Cl expressed in RD2(NL) is in fact CFTR-G_Cl. The presence of an apical amiloride-sensitive G_Na was shown by the amiloride (10⁻⁵ M) inhibition of G_Na as indicated by a reduction of V_t and equivalent short circuit current by 78.0 ± 3.1% and 77.9 ± 2.6%, respectively, and an increase in R_t by 7.2 ± 0.8%,n=36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-G_Cl is expressed in a purely absorptive tissue. It provides an opportunity to study the properties and role of CFTR in the context of absorptive function in immortalized epithelial cells.     

Effect of of ATP on Neurons of the Rat Intact Nodose Ganglion

Under intracellular recording, we studied the effect of ATP on nerve cells of the rat intact nodose ganglion. The resting membrane potential of the examined neurons was, on average, –60.3 ± 1.4 mV (n = 84); among such units, 88% were classified as C cells. Local application of 2 mM ATP to the surface of the ganglion using a modified laminar flow system led to depolarization of neurons by 7.1 ± 0.9 mV, on average (n = 19). A blocker of P2X receptors, PPADS (100 μM), suppressed these depolarization responses, decreasing their amplitude, on average, to 16 ± 3% (n = 3) of the initial value. The obtained data indicate that an overwhelming majority of neurons of the intact nodose ganglion possess functional P2X receptors on their membranes. The absence of the corresponding responses in a considerable part of neurons of intact spinal ganglia [13-15] was, apparently, determined by the fact that P2X receptors in the course of the described experiments had enough time to desensitize before ATP reached the effective concentration.