bluuebunny/crossref_metadata_2025 huggingface dataset:
A voltage-operable current is involved in Ca2+ entry in human lymphocytes whereas ICRAC has no apparent role
Authors: Densmore J. J. , Haverstick D. M. , Szabo G. , Gray L. S.
Published: December 2017

Presently, it is thought that a non-voltage-gated current is responsible for activation-induced Ca2+ entry in nonelectrically excitable cells such as lymphocytes. However, it has also been proposed that the pathway instead involves a second messenger-regulated Ca2+ channel that is voltage operable, where "voltage operable" is defined as an intrinsic property of the channel protein(s) rather than a requirement of normal gating. To evaluate the contribution of these currents to activation-induced Ca2+ influx, each was examined with respect to its ability to account for Ca2+ influx as reported by Ca(2+)-sensitive dyes. We identified a set of reagents, nordihydroguaiaretic acid and various calmodulin inhibitors, that inhibits Ca2+ entry and blocks the voltage-operable current but leaves the non-voltage-gated current unaltered. Further-more, nordihydroguaiaretic acid inhibited Ca(2+)-dependent proliferation of mitogen-activated human peripheral blood mononuclear cells or Jurkat T cells and specifically blocked Ca(2+)-dependent interleukin 2 production by Jurkat T cells to a degree similar to the immunosuppressant drug cyclosporin A. We also identified compounds, amiloride and Mn2+, that block the non-voltage-gated current but have no effect on either the voltage-operable current or Ca2+ entry. Correspondingly, amiloride had no effect on Ca(2+)-dependent proliferation of Jurkat cells. These observations imply that blockade of the non-voltage-gated current does not block either Ca2+ entry or Ca(2+)-dependent lymphocyte proliferation, whereas blockade of the voltage-operable current does. The data suggest that the voltage-operable current may be a mediator of activation-induced Ca2+ entry in lymphocytes.
Nitric oxide and superoxide anion production during endothelial cell proliferation
Authors: Arnal J. F. , Tack I. , Besombes J. P. , Pipy B. , Negre-Salvayre A.
Published: December 2017

We have previously reported that nitric oxide (NO) synthase activity, protein, and mRNA are increased in proliferating compared with postconfluent bovine aortic endothelial cells (BAEC). Because superoxide anion inactivates NO, in the present study, we have assessed the effect of proliferation on superoxide anion production by use of cytochrome c reduction. The superoxide anion production in proliferating cells was increased about threefold compared with postconfluent cells in both basal and calcium ionophore-stimulated conditions and exceeded the amount of released nitrite and nitrate (NOx) in all cases. A-23187 (1 microM) stimulated the superoxide anion production about twofold at all stages of confluence. Because superoxide anion can inactivate NO, we then assessed the effect of proliferation on NO bioactivity released in the conditioned medium, by use of RFL-6 cells (reporter cells very rich in guanylate cyclase, which on activation by NO generates guanosine 3',5'-cyclic monophosphate, second messenger of NO). In the absence of added superoxide dismutase (SOD) in the conditioned medium, the guanylate cyclase-stimulating activities evoked by A-23187 from proliferating and growth-arrested cells were similar, despite a greater NOx release in the former. When SOD (100 U/ml) was added in the conditioned medium, the guanylate cyclase-stimulating activity evoked by 1 microM A-23187 was increased approximately 10-fold and closely paralleled NOx release (i.e., was greater in supernatant of proliferating cells than in that of growth-arrested cells). Thus BAEC release more superoxide anion extracellularly than NO at all stages of confluence. Endothelium-derived superoxide anion is a major determinant of the breakdown of NO.
Evidence for a central role of transcription in the timing mechanism of a circadian clock
Authors: Khalsa S. B. , Whitmore D. , Bogart B. , Block G. D.
Published: December 2017

The retinal circadian clock in the isolated in vitro eye of the marine mollusc Bulla gouldiana exhibits a phase-dependent requirement for transcription. The transcription-sensitive phase extends through most of the subjective day and therefore is substantially longer than the previously reported translation-sensitive phase. Lower concentrations of transcription inhibitors yield a significant dose-dependent lengthening of circadian period. Clock motion can be stopped by a high concentration of the transcription inhibitor 5,6-dichlorobenz-imidazole riboside (DRB) when applied during the sensitive phase; after withdrawal of the inhibitor, motion resumes from the phase at which it was stopped. In a double-pulse experiment, phase shifts to light pulses applied after DRB pulses, and not during the translation-sensitive phase, indicate that the inhibition of transcription has immediate effects on the phase of the clock. These data suggest that DRB-induced phase shifts are independent of translation, which implies that the rate of transcription itself plays a significant role in the mechanism underlying the generation of the circadian cycle.
Caffeine- and carbachol-induced Cl- and cation currents in single opossum esophageal circular muscle cells
Authors: Wang Q. , Akbarali H. I. , Hatakeyama N. , Goyal R. K.
Published: December 2017

Cl- and cation currents may play important roles in esophageal smooth muscle membrane potential changes and contraction. We studied Ca2+ release-activated cell-shortening and membrane currents in single cells freshly dispersed from the circular muscle of the opossum esophagus using the standard patch-clamp whole cell recording method. Caffeine (10-20 microM) and carbachol (10-100 microM) shortened the single smooth muscle cells by releasing intracellular Ca2+. At a holding potential of 0 mV, spontaneous transient outward currents STOCs, representing spontaneous Ca(2+)-activated K+ currents) were recorded. Caffeine, carbachol, or ionomycin evoked large outward currents (up to 1,650 pA) and subsequently abolished STOCs. At a holding potential of -50 mV in K(+)-containing solutions, an outward current in response to the agonists was observed; in some cells, the outward current followed an inward current. In K(+)-free solutions, the agonists induced only an inward current whose reversal potential was shifted by alteration of the anion gradient but not by that of the cation. With a low-Cl- pipette solution (Cl- substituted by glucuronate or glutamate), the inward currents were dependent mainly on the external cation gradient. This cation channel was permeable to Ba2+. Inclusion of 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette solution abolished all these currents. These data suggest that in the opossum esophageal circular muscle 1) Ca2+ released from the intracellular stores by caffeine and carbachol is sufficient to induce single smooth muscle cell contraction and 2) the caffeine-, carbachol-, and ionomycin-induced membrane currents consist of Ca(2+)-activated K+, Cl-, and cation conductances.
Connexin 32/38 chimeras suggest a role for the second half of inner loop in gap junction gating by low pH
Authors: Wang X. G. , Peracchia C.
Published: December 2017

Gap junction channels are regulated by gates that close with cytosolic acidification and transjunctional voltage (Vj). For identifying the connexin (Cx) domain(s) involved in channel gating, CO2 and Vj sensitivities of channels made of Cx38, Cx32, Cx32/Cx38 chimeras, and Cx32 mutants were studied in Xenopus oocyte pairs. Recently, we have reported that Cx38 is more sensitive to CO2 and Vj than Cx32 because of differences in the Cx inner loop. To identify the responsible inner loop domain, chimeras of Cx32/Cx38 in which the first (I1) or the second (I2) half of the inner loop of Cx38 replaced that of Cx32 and I2 mutants of Cx32 were tested. The chimera Cx32/Cx38I2 (Cx32 with I2 of Cx38) was like Cx38 in CO2 sensitivity but like Cx32 in Vj sensitivity. Cx32/Cx38I1 (Cx32 with I1 of Cx38) did not express channels. Of the three Cx32 mutants, Cx32-VH/IR VH of Cx32 replaced with IR of Cx38) and Cx32-WW/MC WW of Cx32 replaced with MC of Cx38) were like Cx32 in both CO2 and Vj sensitivity, whereas Cx32-S*T/Q*P (S*T of Cx32 replaced with Q*P of Cx38) was closer to Cx38 in CO2 sensitivity but behaved like Cx32 in Vj gating. The data suggest that I1 and I2 contain domains relevant for Vj and CO2 gating, respectively.
Na+/Ca2+ exchange currents and SR Ca2+ contents in postinfarction myocytes
Authors: Zhang X. Q. , Tillotson D. L. , Moore R. L. , Zelis R. , Cheung J. Y.
Published: December 2017

Myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) had lower peak cytosolic free Ca2+ concentration ([Ca2+]i) and reduced maximal extent of cell shortening during contraction, but Ca2+ entry via L-type Ca2+ channels was normal. In the current study using whole cell patch-clamp technique, reverse Na+/Ca2+ exchange current (INa/Ca; 3 Na+ out:1 Ca2+ in) was measured in myocytes in which Na+, K+, and Ca2+ currents were blocked or minimized. Steady-state outward currents measured under these conditions increased with depolarization or with elevation of extracellular Ca2+ concentration ([Ca2+]o) from 1.8 to 5.0 mM, but were inhibited by 5 mM Ni2+ or by reduction of [Ca2+]i to near zero. In addition, reduction of cytosolic free Na+ concentration or of [Ca2+]i also decreased the amplitude of the outward current. These characteristics indicate the outward current was INa/Ca operating in reverse mode. Reverse INa/Ca was significantly lower in MI myocytes, especially at more positive voltages. In addition, sarcoplasmic reticulum (SR)-releasable Ca2+ content as estimated by integrating forward INa/Ca during caffeine-induced SR Ca2+ release was also significantly lower in MI myocytes. Depressed Na+/Ca2+ exchange activity may contribute to abnormal [Ca2+]i dynamics in MI myocytes.
Study of nonspecific cation channel coupled to P2z purinergic receptors using an acid load technique
Authors: Lachish M. , Alzola E. , Chaib N. , Metioui M. , Grosfils K. , Kabre E. , Moran A. , Marino A. , Dehaye J. P.
Published: December 2017

The intracellular pH (pHi) of rat submandibular cells was measured by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The cells recovered from ammonium (30 mM) prepulse to their resting pHi within 10 min. Ethylisopropylamiloride (EIPA), an inhibitor of the Na+/H+ exchanger, slows the rate of pHi recovery. ATP (1 mM), in the presence of EIPA, increases the rate of recovery 3.7-fold in the absence or presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The recovery was blocked by the addition of 5 mM Mg2+ or 10 microM Coomassie blue. The response was elicited by 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate but not by ADP, UTP, adenyl (beta-gamma-methylene)-diphosphonate, 2-methylthioadenosine 5'-triphosphate, or muscarinic or beta-adrenergic agonists. The purinergic response was also observed when the cells were acidified by sodium propionate and could not be mimicked by the depolarization of the plasma membrane. Aluminum fluoride did not reproduce the response to ATP, suggesting that the observed response does not involve a high-molecular-weight GTP-binding protein. It is concluded that the activation of P2z receptors, probably by the opening of nonspecific cation channels, increases the permeability to protons in rat submandibular glands.
Both M1 and M3 receptors regulate exocrine secretion by mucous acini
Authors: Culp D. J. , Luo W. , Richardson L. A. , Watson G. E. , Latchney L. R.
Published: December 2017

We investigated the role of M1 and M3 receptors in regulating exocrine secretion from acini isolated from rat sublingual glands. In secretion experiments, we derived affinity values (KB) from Schild regression analysis for the antagonists pirenzepine (61.0 nM) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 1.06 nM). The KB for 4-DAMP is similar to its affinity value [equilibrium dissociation constant from competition studies (Ki); 1.81 nM] determined from radioligand competition experiments. In contrast, the KB for pirenzepine is between its high-affinity (17.6 nM) and low-affinity (404 nM) Ki values. In separate secretion experiments, we found that the M1 receptor antagonist, M1-toxin, induces a rightward shift in the concentration-response curve to muscarinic agonist and inhibits maximal secretion by 40%. The inhibitory effect of M1-toxin appears specific for M1 receptor blockade, since the toxin abolishes acinar high-affinity pirenzepine-binding sites and does not inhibit secretion induced by nonmuscarinic agents. Additional pharmacological studies indicate muscarinic receptors do not function through putative neural elements within isolated acini. Our combined results are consistent with both M1 and M3 receptors directly regulating mucous acinar exocrine secretion and indicate M3 receptors alone are insufficient to induce a maximal muscarinic response.
Myoglobin content and oxygen diffusion: model analysis of horse and steer muscle
Authors: Conley K. E. , Jones C.
Published: December 2017

We test the hypothesis that myoglobin is important for O2 supply near the oxidative capacity of muscle. This hypothesis is evaluated with a simple model that incorporates the properties of heart and skeletal muscle tissue taken from steers and horses exercising at their maximum O2 consumption rate. These tissue samples allowed us to set the bounds on oxidative demand and O2 flux from red blood cells to the core of the muscle fiber, to estimate the blood and tissue capacities for O2 diffusion, and to define the capillary blood PO2 driving this O2 flux. A model combining blood convection with tissue diffusion indicates that O2 diffusion alone is insufficient to achieve the measured O2 fluxes in many samples. The myoglobin content of these fibers is significantly correlated with this O2 diffusion limitation and provides sufficient additional O2 flux to meet muscle O2 demand. The presence of myoglobin maintains the PO2 in the fiber core above anoxic levels for the majority of muscles. Thus myoglobin is critical to O2 supply at fluxes near the maximum and prevents anoxia by maintaining PO2 above levels needed to support mitochondrial function.
Altered contractility of urinary bladder in diabetic rabbits: relationship to reduced Na+ pump activity
Authors: Gupta S. , Yang S. , Cohen R. A. , Krane R. J. , Saenz De Tejada I.
Published: December 2017

We studied the effect of alloxan-induced diabetes on Na+ pump activity in isolated rabbit bladder strips. In addition, the effects of diabetes and the Na+ pump inhibitor ouabain on contractions induced by carbachol (CCh) and KCl were studied. In bladder strips from diabetic rabbits, ouabain-sensitive 86Rb+ uptake (a measure of Na+ pump activity) was approximately 50% less compared with strips from normal bladder. Diabetes also reduced the maximum contractions induced by CCh and KCl. Treatment of bladder strips with ouabain alone caused an acute concentration-dependent increase in tone. In contrast, longer incubation with ouabain inhibited CCh- and KCl-induced contractions in normal and diabetic bladders. Furthermore, differences in agonist-mediated contractions observed between normal and diabetic bladders were abolished in the presence of the maximally effective concentration of ouabain (10 microM). The ability of CCh to cause contraction in normal and diabetic rabbit bladders was also significantly inhibited by the Na+ ionophore monensin but not by the Ca2+ ionophore A-23187 or by depolarization with KCl. Monensin also inhibited KCl-induced contractions in normal bladder strips. These results indicate that 1) Na+ pump activity is an important modulator of bladder smooth muscle tone, 2) diabetes diminishes Na+ pump activity and inhibits agonist-induced contractions in bladder, and 3) an increase in intracellular Na+ concentration, secondary to inhibition of bladder smooth muscle Na+ pump activity, is associated with reduced responsiveness to contractile agonists. Diminished Na+ pump activity in diabetes may, in part, contribute to the development of bladder cystopathy.
Modulation of CFTR chloride channels by calyculin A and genistein
Authors: Yang I. C. , Cheng T. H. , Wang F. , Price E. M. , Hwang T. C.
Published: December 2017

Modulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by calyculin A and genistein was studied in Hi-5 insect cells infected with baculovirus containing the wild-type CFTR cDNA. In cell-attached patches, CFTR channel activity was not observed until stimulated by forskolin in 90% of the cells, suggesting a low level of basal adenosine 3',5'-cyclic monophosphate activity. Calyculin A, a specific inhibitor of phosphatases 1 and 2A, increased forskolin-induced CFTR activity by 17.2-fold. CFTR channel currents did not deactivate completely after forskolin was withdrawn in the continued presence of calyculin A. Genistein enhanced forskolin-induced CFTR activity by 44.9-fold but could neither activate the CFTR by itself nor prevent complete deactivation on removal of forskolin. Genistein together with calyculin A could adequately prevent deactivation of CFTR currents. Noise analysis of the macroscopic CFTR currents revealed significant differences in the mean current-variance-relationship and the corner frequency of the noise spectra between currents activated by forskolin plus genistein and those activated by forskolin plus calyculin A. Furthermore, genistein enhanced CFTR activity induced by saturating concentrations of forskolin and calyculin A. Our results suggest that genistein and calyculin A modulate the CFTR by different mechanisms and that genistein might inhibit calyculin A-insensitive dephosphorylation of the CFTR.
Effects of F-actin stabilization or disassembly on epithelial Cl- secretion and Na-K-2Cl cotransport
Authors: Matthews J. B. , Smith J. A. , Hrnjez B. J.
Published: December 2017

Previous studies showed that cAMP-dependent transepithelial Cl- secretion of the intestinal cell line T84 is reduced by the F-actin stabilizer phalloidin, an effect in part attributable to inhibition of basolateral Na-K-2Cl cotransport. However, secretory responses are preserved in cells treated with the microfilament disrupter cytochalasin D. We explored the effects of cytochalasin D and two novel compounds derived from marine sponges on the Cl- secretory apparatus of T84 cells. Jasplakinolide (which stabilizes F-actin inhibited cAMP-dependent secretion and Na-K-2Cl cotransport. Latrunculin A (which sequesters G-actin monomers) profoundly altered the distribution of F-actin and reduced basal transepithelial resistance with minimal effect on secretion. Cytochalasin D, but not latrunculin A, activated Na-K-2Cl cotransport. The results provide further evidence that vectorial ion transport is influenced by the cytoskeleton and support a model in which disassembly of F-actin by specific pharmacological means or in response to secretory agonists favors activation of Na-K-2Cl cotransport.
Fast and slow skeletal muscles express a common basic profile of acetylcholinesterase molecular forms
Authors: Boudreau-Lariviere C. , Gisiger V. , Michel R. N. , Hubatsch D. A. , Jasmin B. J.
Published: December 2017

Recent evidence suggests that the high content of acetylcholinesterase (AChE) globular form G4, characteristic of fast muscles, is controlled by phasic high-frequency activity performed by these muscles. This indicates that inactive, though still innervated, fast muscles should be devoid of their characteristic G4 pool. Accordingly, in the absence of phasic activity, both fast and slow muscles should exhibit a common basic profile of AChE molecular forms of the slow type. We first tested this hypothesis by examining the AChE content in cultures of myotubes obtained from the fusion of satellite cells originating from fast and slow muscles. These two cell populations produced AChE molecular-form profiles of the slow type characterized by modest levels of G4 together with an increased proportion of the asymmetric forms A8 relative to A12. Second, we determined the impact of muscle paralysis on the specific content of AChE molecular forms of adult rat fast and slow muscles. Complete paralysis of hindlimb muscles was achieved by chronic superfusion of tetrodotoxin (TTX) onto the sciatic nerve. Ten days after TTX inactivation, the distributions of AChE molecular forms of both fast extensor digitorum longus (EDL) and plantaris muscles were transformed into ones resembling the slow soleus, the latter showing no significant modifications in its AChE profile. Finally, we investigated the impact of nerve-mediated phasic high-frequency stimulation of TTX-inactivated fast and slow muscles on the content of AChE molecular forms. This stimulation produced a profile of AChE molecular forms similar to that observed in control EDL muscles, indicating that phasic activation counteracted the TTX-induced transformation in the distribution of AChE molecular forms in fast EDL muscles. Together, these results are consistent with the proposal that adult fast muscles constitutively express a basic profile of AChE molecular forms of the type displayed by slow muscles, onto which varying levels of G4 are added according to the amount of phasic activity performed by the muscles.
beta-Adrenergic modulation of Ba2+ currents and K+ contractures in frog slow skeletal muscle fibers
Authors: Huerta M. , Trujillo X. , Vasquez C.
Published: December 2017

beta-Adrenergic modulation of the Ba2+ current (IBa) and K+ contracture in slow skeletal muscle fibers of the frog (Rana pipiens) were investigated in intact fibers with the three-microelectrode voltage-clamp technique and isometric tension measurements. Application of epinephrine (10(-6) to 10(-5) M) to the bath increased the amplitude of IBa. This increase was blocked by the beta-antagonist propranolol (3 microM), and a similar increase was observed with the beta-specific agonist isoproterenol (1 microM). Thus the epinephrine effect was mediated mainly by beta-adrenergic receptors. External application of permeable 8-bromoadenosine 3',5'-cyclic monophosphate (0.5 mM) increased the amplitude of both IBa and K+ contractures. The present results suggest that beta-adrenergic modulation of IBa in slow skeletal muscle fibers could reflect a modulation of Ca2+ channels via adenosine 3',5'-cyclic monophosphate (cAMP). cAMP (0.5 mM) also potentiated the K(+)-evoked tension in these slow fibers. The physiological contribution made by the modulation of slow skeletal muscle Ca2+ channels to the increase in tension is still not completely understood.
Dual inhibitory effects of dopamine on Na+ homeostasis in rat aorta smooth muscle cells
Authors: Borin M. L.
Published: December 2017

Dopamine is an essential catecholamine, which acts not only as a neurotransmitter in sympathetic neurons but also exhibits vasodilating and natriuretic effects in renal tubular cells, blood vessels, etc. This study describes the effect of dopamine on Na+ influx and Na+ efflux and the resulting changes in intracellular Na+ concentration ([Na+]i). [Na+]i was measured in primary cultured vascular smooth muscle cells from rat aorta with digital imaging of cells loaded with the Na+-sensitive fluorescent indicator, SBFI. Na+ influx and Na+ efflux were measured as changes in [Na+]i under the conditions of inhibition of the Na+ flux in the opposite direction. Dopamine inhibited Na+ influx in a dose-dependent manner with a maximal inhibition, approximately 45%, achieved at 10(-4) M. This effect of dopamine, as suggested by several lines of evidence, was mediated by inhibition of Na+/H+ exchange. Besides inhibition of Na+ efflux, dopamine also, with a similar potency, inhibited Na+ efflux. The latter effect was due to inhibition of the Na+ pump-mediated component of Na+ efflux, since it was not observed when Na+ pump was inhibited. Inhibition of the Na+ pump by dopamine was due to the reduction in its maximal flux and not due to the decrease in the Na+ sensitivity of the pump. Similar to dopamine, activation of protein kinase A by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) caused inhibition of both Na+ influx and Na+ pump-mediated Na+ efflux. In contrast, activation of protein kinase C by the phorbol ester, phorbol 12,13-dibutyrate, caused activation of both Na+ influx and Na+ pump-mediated Na+ efflux. H-7, a nonspecific protein kinase inhibitor, abolished the inhibitory effects of either dopamine or 8-BrcAMP on Na+ efflux but did not affect the inhibitory effects of these compounds on Na+ influx. Dopamine either did not change [Na+]i or evoked a slight, 2-3 mM, increase in [Na+]i. Together, these results demonstrate that, in rat aortic smooth muscle cells, 1) dopamine inhibits Na+/H+ exchange-mediated Na+ influx, 2) dopamine inhibits Na+ pump-mediated Na+ efflux, 3) these effects of dopamine are mediated by an increase in cellular cAMP and, at least in the case of inhibition of the Na+ efflux, by the activation of protein kinase A, and 4) dopamine causes either small or no changes in [Na+]i, due to almost equal inhibition of Na+ influx and Na+ efflux.
GTP gammaS removal of D-600 block of skeletal muscle excitation-contraction coupling
Authors: Carney-Anderson L. , Thompson L. V. , Huetteman D. A. , Donaldson S. K.
Published: December 2017

G proteins interacting with dihydropyridine receptors (DHPR) in transverse tubules (TT) of skeletal muscle may have a role in skeletal excitation-contraction (EC) coupling. The aim of this study was to determine the effects of G protein-specific nucleotides [guanosine 5'-O-(3-thiotriphosphate) (GTP gammaS) and guanosine 5'-O-(2-thiodiphosphate) (GDP betaS)] on the EC coupling mechanism in the presence of D-600, an agent that blocks EC coupling by immobilizing the voltage-sensing subunit of the DHPR in its inactivated state. By use of the mechanically peeled single-fiber preparation from rabbit adductor magnus skeletal muscle, 50 microM GTP gammaS and 500 microM GDP betaS were applied with the fiber in a D-600-induced state of blocked EC coupling. Neither nucleotide served as an independent stimulus for sarcoplasmic reticulum (SR) Ca2+ release when added to the TT polarizing bath under conditions of D-600 block. The presence of GTP gammaS or GDP betaS during a complete EC coupling cycle removed the D-600 block of EC coupling, despite continuous bath D-600. After the nucleotides were washed out, in the continued presence of D-600, the D-600 block of EC coupling was reestablished. In contrast, GTP gammaS added only during the period of TT depolarization under D-600 block did not remove the D-600 block of EC coupling, even though GTP gammaS did stimulate SR Ca2+ release. GTP gammaS had no effect on submaximum (0.5-1.0 mM) caffeine contractures and thus is unlikely to be acting through the Ca2+-induced Ca2+ release mechanism of the SR. These data suggest that the molecular binding site for GTP gammaS and GDP betaS is likely to be in the TT near the DHPR, perhaps on a G protein.
Effects of pH on the potassium current in rabbit corneal epithelial cells
Authors: Rich A. , Bartling C. , Farrugia G. , Rae J. L.
Published: December 2017

The effects of pH on K+ conductance were measured using the amphotericin perforated-patch whole cell voltage-clamp technique in freshly dispersed rabbit corneal epithelial cells. Bath perfusion with pH 6.00 Ringer solution after standard Ringer solution (pH 7.35) increased outward K+-selective current (I(K)) from 120 +/- 29 to 312 +/- 64 pA during a step depolarization to +50 mV and hyperpolarized the resting membrane potential (Em) from -52 +/- 5 to -62 +/- 3 mV (n = 15, P < or = 0.05). Increasing bath pH to 8.5 decreased I(K) from 183 +/- 40 to 114 +/- 35 pA (n = 6, P < or = 0.05) and depolarized Em from -63 +/- 6 to -53 +/- 5 mV (n = 6, P < or = 0.05). Intracellular acidification using the weak electrolyte (NH4)2SO4 also increased I(K) from 83 +/- 15 to 183 +/- 20 pA (n = 4, P = 0.01) and hyperpolarized Em from -51 +/- 8 to -68 +/- 6 mV (P = 0.002). Intracellular alkalinization reduced I(K) to 66 +/- 10 pA and depolarized Em to -36 +/- 8 mV (P = 0.009). Single channel studies in perforated outside-out vesicles showed that a decrease in bath pH from 7.35 to 6.00 was accompanied by an increase in the single channel open probability (NPo) from 0.43 to 0.64 at an Em of 15 mV. NPo was also increased in cell-attached patches. The unitary conductance, measured from -100 to +100 mV, was not changed. These results indicate that pH modulates I(K) in rabbit corneal epithelial cells by changes in NPo.
Muscarinic stimulation of tracheal smooth muscle cells activates large-conductance Ca2+-dependent K+ channel
Authors: Wade G. R. , Sims S. M.
Published: December 2017

Pages C658-C665: G. R. Wade and S. M. Sims. “Muscarinic stimulation of tracheal smooth muscle cells activates large-conductance Ca2+-dependent K+ channel.” Page C662, Fig. 6: the second half of the trace in A was inadvertently duplicated from B. The revised Fig. 6 below shows the correct channel traces. We wish to emphasize that the scientific point of the figure, the reversible antagonism of the cholinergic response by atropine, as well as the quantification in D, remains sound. The data were filtered at 400 Hz and sampled off-line from digital videotape at 2 kHz.

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Effects of extracellular ATP on ion transport and [Ca2+]i in Mongolian gerbil middle ear epithelium
Authors: Furukawa M. , Ikeda K. , Yamaya M. , Oshima T. , Sasaki H. , Takasaka T.
Published: December 2017

The present study investigates the effects of ATP on ion transport and Ca2+ mobilization in primary cultures of gerbil middle ear epithelium. Extracellular ATP and other nucleotides elicited a transient increase in short-circuit current (I(sc)) in a dose-dependent manner. The rank order of potency in I(sc) responses corresponded with the sequence of the P(2u) purinergic receptor when applied to the mucosal or the basolateral side. Extracellular ATP induced a rise in the intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. A similar rank order of potency in the ATP-induced increases in [Ca2+]i was observed. The time courses of the [Ca2+]i responses induced by ATP closely paralleled those of the I(sc) responses. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester greatly reduced the increase in the I(sc) and [Ca2+]i responses to ATP. Diphenylamine-2-carboxylate, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, quinidine, and Ba2+ significantly blocked the ATP-stimulated I(sc). Glibenclamide had no effect on the ATP-stimulated I(sc). These results are consistent with the hypothesis that extracellular ATP and other nucleotides induce Cl- secretion across the apical membrane in cultured gerbil middle ear epithelium by activation of the apical Cl- channels via multiple modes of regulation.
Characterization of renin activity in brown adipose tissue
Authors: Shenoy U. , Cassis L.
Published: December 2017

Angiotensin (ANG) II plays a vital role in blood pressure regulation and body fluid homeostasis. Although many peripheral tissues synthesize components of the renin-ANG system, very few synthesize all of the major components involved in the generation ofANG II. This study used interscapular brown adipose tissue (ISBAT) as a model system to evaluate the mechanism of ANG II generation in an extrarenal tissue. Polymerase chain reaction analysis of DNA from ISBAT demonstrated angiotensinogen gene expression; however, renin gene expression was not detected. Renin activity that was not completely derived from the residual blood pool was detected in ISBAT homogenates. Kinetic parameters for renin activity were similar in ISBAT and adrenal gland. Renin activity was partially inhibited by anti-renin antibody and completely inhibited by a specific rat renin inhibitor. Bilateral nephrectomy did not decrease renin activity in ISBAT. Western blot analysis, employing two species-specific renin antibodies, indicated the presence of a variety of isoforms of renin in ISBAT. The presence of renin activity in isolated brown adipocytes demonstrated that the enzyme is localized to adipocytes. The release of immunoreactive ANG peptides from ISBAT slices over 3 h indicated de novo synthesis. These studies support the existence of a local renin-ANG system in ISBAT and suggest involvement of renin in the formation of ANG II.
Chondrocytes in the endochondral growth cartilage are not hypoxic
Authors: Shapiro I. M. , Mansfield K. D. , Evans S. M. , Lord E. M. , Koch C. J.
Published: December 2017

From an anatomic viewpoint, the blood supply to chondrocytes in the growth cartilage is limited. As a result, it has been suggested that these cells are hypoxic and that this condition regulates chondrocyte maturation and cartilage mineralization. We examined the state of chondrocyte oxygenation in the chick growth plate using a hypoxia-sensing drug, EF5. EF5 is a pentafluorinated derivative of the 2-nitroimidazole, etanidazole, that is metabolically reduced by oxygen-inhibitable nitroreductase(s). Reduced EF5 covalently forms adducts with cellular macromolecules that can be visualized with a highly specific fluorochrome-conjugated monoclonal antibody. When EF5 was injected into chicks and tissues were subsequently examined by immunohistochemical techniques, chondrocytes in articular, proliferating, and hypertrophic cartilage exhibited a low level of fluorescence-detectable binding, suggesting the absence of significant hypoxia. We confirmed that the results were not confounded by tissue-specific factors related to low-chondrocyte nitroreductase activity or problems from drug diffusion into cartilage. Using in vitro systems, we showed that, under conditions of imposed hypoxia, EF5 diffused into the tissue and was bound to chondrocytes. With the use of an in vivo model in which hypoxia was artificially induced by death, chick chondrocytes were found to bind the drug. Although the EF5-binding method is not optimally suited for determining the precise oxygen partial pressure in heterogeneous tissues, such as the growth plate, we concluded that chick chondrocytes are not oxygen deficient in vivo.
Modulation of maxi-K+ channels by voltage-dependent Ca2+ channels and methacholine in single airway myocytes
Authors: Wang Y. X. , Fleischmann B. K. , Kotlikoff M. I.
Published: December 2017

The role of Ca2+ influx through voltage-dependent Ca2+ channels and the inhibitory effects of methacholine on large-conductance Ca2+-activated K+ (K(Ca)) channels (maxi-K+ channels) were studied in voltage-clamped (nystatin), fura 2-loaded airway smooth muscle cells. Spontaneous transient outward currents (STOCs) were strongly coupled to voltage-dependent Ca2+ channel activity; activity was suppressed by nisoldipine and Cd2+ and increased by BAY K 8644 within seconds. Moreover, release of intracellular Ca2+ by caffeine or cyclopiazonic acid only partially suppressed STOCs, and the remainder were almost completely blocked by nisoldipine. Methacholine suppressed STOCs but also significantly decreased the mean outward current. Whole cell current inhibition was observed in the presence of 4-aminopyridine but not in the presence of charybdotoxin. Caffeine inhibited STOCs but macroscopic outward currents were not altered. In the continued presence of caffeine, methacholine abolished the remaining STOCs and decreased the mean K+ current. We conclude that STOCs are activated by influx of Ca2+ through plasmalemmal voltage-dependent Ca2+ channels, as well as by release of Ca2+ from intracellular stores, and muscarinic stimulation depresses the mean K(Ca) current via a pathway independent of the depletion of intracellular Ca2+ stores.
Ins(3,4,5,6)P4 specifically inhibits a receptor-mediated Ca2+-dependent Cl- current in CFPAC-1 cells
Authors: Ho M. W. , Shears S. B. , Bruzik K. S. , Duszyk M. , French A. S.
Published: December 2017

We have examined the role of inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] in the control of Cl- current in CFPAC-1 cells. Intracellular Ins(3,4,5,6)P4 had no effect on basal current, but it produced a five- to sevenfold reduction in the Cl- current stimulated by either 2 microM extracellular ATP or by 1 microM extracellular thapsigargin. The half-maximally effective dose of Ins(3,4,5,6)P4 was 2.9 microM, and 4 microM blocked >80% of the ATP-activated current. In contrast, 10 microM Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, or Ins(1,3,4,6)P4 enhanced rather than inhibited the ATP-activated Cl- current, although Ins(1,4,5,6)P4 only acted transiently. These stimulatory effects were Ca2+ dependent and largely inhibited by coapplication of equimolar Ins(3,4,5,6)P4. Inositol 1,3,4,5,6-pentakisphosphate, the precursor of Ins(3,4,5,6)P4, did not affect Cl- current. These data consolidate and extend the hypothesis that Ins(3,4,5,6)P4 is an important intracellular regulator of Cl- current in epithelial cells.
Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat
Authors: Quednau B. D. , Nicoll D. A. , Philipson K. D.
Published: December 2017

The gene coding for the Na+/Ca2+ exchanger NCX1 is characterized by a cluster of six exons (A, B, C, D, E, and F) coding for a variable region in the COOH terminus of the large intracellular loop of the protein. Alternative splicing of these exons generates multiple tissue-specific variants of NCX1. Using reverse transcriptase-polymerase chain reaction, we analyzed eight previously described and four new splicing isoforms of NCX1 in a wide variety of tissues and cells. Exons A and B are mutually exclusive, as shown in earlier studies, and splicing isoforms containing exon A are preferentially expressed in heart, brain, and skeletal muscle, whereas splicing variants with exon B are found in all rat tissues except heart. The second and third isoforms of the Na+/Ca2+ exchanger, NCX2 and NCX3, show a deletion of 37 amino acids in the intracellular loop corresponding to parts of the variable region of NCX1. We identified three splicing isoforms of NCX3 in brain and skeletal muscle by reverse transcriptase-polymerase chain reaction. These splice variants are generated by including either of two alternative exons equivalent to the NCX1 exon A or B and by including or excluding a sequence equivalent to the NCX1 exon C. We did not detect any alternative splicing of NCX2. We examined selected tissues from neonatal and adult rats and found developmental regulation for NCX1 and NCX3 splicing isoforms in skeletal muscle. Specific isoform patterns were also detected for NCX1 and NCX3 in cultured cortical neurons, astrocytes, and oligodendrocytes. We suggest a new terminology to distinguish the different splice variants of individual NCX isoforms.
Role of extracellular signal-regulated kinase and PKC alpha in cytosolic PLA2 activation by bradykinin in MDCK-D1 cells
Authors: Xing M. , Tao L. , Insel P. A.
Published: December 2017

The actions of bradykinin (BK) in Madin-Darby canine kidney (MDCK) and other cell types involve formation of arachidonic acid (AA) and AA products by as-yet-undefined mechanisms. We found that BK promoted AA release and an increase in phospholipase A2 (PLA2) activity in subsequently prepared MDCK-D1 cell lysates, both of which were Ca2+ dependent and were inhibited by the 85-kDa cytosolic PLA2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone. In addition, BK treatment of cells led to increased PLA2 activity of cPLA2 immunoprecipitated from lysates. Thus BK receptors mediate AA release via cPLA2 in MDCK-D1 cells. The BK-promoted increase of cPLA2 activity was reversed by treatment of cell lysates with potato acid phosphatase, implying that phosphorylation underlies the activation of cPLA2. However, extracellular signal-regulated kinase (ERK) appeared not to be responsible for this phosphorylation, because treatment of cells with BK (in contrast with the results obtained with epinephrine and phorbol ester) caused neither enzyme activation nor phosphorylation (as judged by molecular mass shift) of this kinase. Although the alpha isoform of protein kinase C (PKC alpha) is responsible for AA release promoted by phorbol ester treatment of MDCK-D1 cells (C. Godson, K.S. Bell, and P.A. Insel. [corrected] J. Biol. Chem. 268: 11946-11950, 1993), neither treatment of cells with the PKC alpha-selective inhibitor GF109203X nor transfection of cells with PKC alpha antisense cDNA altered BK-mediated AA release. We conclude that PKC alpha is unlikely to play an important role in the regulation of cPLA2 by BK receptors in MDCK-D1 cells. The tyrosine kinase inhibitor herbimycin A, on the other hand, inhibited both BK-promoted AA release in intact cells and cPLA2 activation in cell lysates, suggesting the involvement of tyrosine kinase in the regulation of this lipase by BK receptors. Taken together, these data suggest that BK receptors in MDCK-D1 cells regulate cPLA2 via phosphorylation mediated by kinases other than ERK and PKC alpha.
ET-1 cooperates with EGF to induce mitogenesis via a PTX-sensitive pathway in airway smooth muscle cells
Authors: Fujitani Y. , Bertrand C.
Published: December 2017

We have examined the mitogenic effect of endothelin-1 (ET-1) alone or in combination with epidermal growth factor (EGF) in cultured airway smooth muscle cells (ASM) from guinea pig. ET-1 showed a weak mitogenic activity compared with the effect of EGF. However, when ET-1 and EGF were applied simultaneously, ET-1 synergistically enhanced the mitogenic activity of EGF. Neither inhibition of phospholipase C-beta nor depletion of protein kinase C affected this synergism. On the other hand, pertussis toxin (PTX), a Gi protein inhibitor, abolished the synergistic effect of ET-1 on EGF-induced mitogenesis. ET-1 induced a transient mitogen-activated protein (MAP) kinase activation peaking at 5 min. In contrast, EGF induced a stronger signal that was maintained for up to 20 min. However, concomitant stimulation of ASM with ET-1 and EGF caused an enhanced MAP kinase activation compared with EGF alone. Moreover, PTX abolished the enhanced MAP kinase activation observed in this condition. These results indicate that ET-1 can interact with an EGF-induced mitogenic axis through the Gi protein-dependent pathway, which is distinct from its direct mitogenic pathway.
Glutathione content of V79 cells in two- or three-dimensional culture
Authors: Romero F. J. , Zukowski D. , Mueller-Klieser W.
Published: December 2017

The cellular glutathione (GSH) content of two- and three-dimensional cell cultures of V79 hamster lung cells has been studied. As previously described, cells in monolayer cultures show a decrease in GSH when they reach the confluent state. Three-dimensional cell cultures (multicell spheroids) allow a smoother transition from the initial proliferating to the nonproliferating status, and they show a central area of necrosis when a certain diameter is reached. Cellular GSH content in spheroids is variable throughout the culturing period: 1) GSH content (expressed per mg protein) is lower in spheroids with central necrotic areas than in smaller spheroids without necrosis, and 2) results expressed per cell number show a sharp increase around the diameter where necrosis appears. Once a relatively large necrotic area has been established, GSH decreases again to approximately the prenecrotic level. Interestingly, this GSH "peak" is not dependent on the time in culture but on the spheroid size. Acute hypoxia occurs in central areas of spheroids at a much higher size range than those described herein. Thus we suggest a combination of factors, which may include oxidative stress among others, as the explanation for these cellular GSH variations.
Glucose transport by isolated plasma membranes of the bovine blood-brain barrier
Authors: Lee W. J. , Peterson D. R. , Sukowski E. J. , Hawkins R. A.
Published: December 2017

Luminal and abluminal endothelial plasma membrane vesicles were isolated from bovine cerebral microvessels, the site of the blood-brain barrier. Glucose transport across each membrane was measured using a rapid-filtration technique. Glucose transport into luminal vesicles occurred by a stereospecific energy-independent transporter [Michaelis-Menten constant (K(m)) = 10.3 +/- 2.8 (SE) mM and maximal velocity (Vmax) = 8.6 +/- 2.0 nmol.mg protein(-1).min-1]. Kinetic analysis of abluminal vesicles also showed a transport system with characteristics similar to the luminal transporter (K(m) = 12.5 +/- 2.3 mM and Vmax = 10.0 +/- 1.0 nmol.mg protein-1.min-1). These functional, facilitative glucose transporters were symmetrically distributed between the luminal and abluminal membrane domains, providing a mechanism for glucose movement between blood and brain. The studies also revealed a Na-dependent transporter on the abluminal membrane with a higher affinity and lower capacity than the facilitative transporters (K(m) = 130 +/- 20 microM and Vmax = 1.59 +/- 0.44 nmol.mg protein-1.min-1. The abluminal Na-dependent glucose transporter is in a position to transport glucose from the brain extracellular fluid into the endothelial cells of the blood-brain barrier. The functional significance of its presence there remains to be determined.
Regulation of inducible and neuronal nitric oxide synthase gene expression by interferon-gamma and VIP
Authors: Bandyopadhyay A. , Chakder S. , Rattan S.
Published: December 2017

The studies examined the regulation of inducible and neuronal nitric oxide synthases (iNOS and nNOS, respectively) in the rat brain, stomach, rectum, and spleen. Relative expression of iNOS and nNOS mRNAs was quantified by the sensitive method of polymerase amplification reactions. The NOS proteins were determined by Western blot, using specific antibodies. Highest levels of nNOS and iNOS mRNAs were expressed in the rat brain and spleen, respectively. Furthermore, both nNOS and iNOS were expressed in the stomach and rectum. Interestingly, treatment of tissues with lipopolysaccharides or cytokine interferon-gamma (IFN-gamma) induced the expression of iNOS and decreased that of nNOS, representing a shift from one isoform to the other. When the tissues were treated with IFN-gamma followed by vasoactive intestinal polypeptide (VIP), the induction of iNOS was reduced by VIP. The changes in iNOS and nNOS expression at the transcriptional level corresponded to those at the translational level. The data suggest a regulatory role of IFN-gamma and VIP in the expression iNOS and nNOS and a counterregulation of iNOS and nNOS in rat tissues.
Protein prenylation is required for aldosterone-stimulated Na+ transport
Authors: Blazer-Yost B. L. , Hughes C. L. , Nolan P. L.
Published: December 2017

Aldosterone stimulation of transcellular Na+ flux in polarized epithelial cells is dependent on at least one transmethylation reaction, but the substrate of this signaling step is unknown. Because it is clear that the majority of cellular protein methylation occurs in conjunction with protein prenylation, we examined the importance of prenylation to aldosterone-stimulated Na+ transport in the A6 cell line. Lovastatin, an inhibitor of the first committed step of the mevalonate pathway, inhibits the natriferic effect of aldosterone but does not inhibit insulin-stimulated Na+ flux. The addition of a farnesyl group does not appear to be involved in aldosterone's action. Neither alpha-hydroxyfarne-sylphosphonic acid, an inhibitor of farnesyl:protein transferase, nor N-acetyl-S-farnesyl-L-cysteine, an inhibitor of farnesylated protein methylation, inhibits the hormone-induced increase in Na+ transport. In contrast, N-acetyl-S-geranyl-geranyl-L-cysteine, an inhibitor of geranylgeranyl protein methylation, completely abolishes the aldosterone-induced increase in Na+ flux with no effect on insulin-mediated Na+ transport or cellular protein content. These data indicate that methylation of a geranylgeranylated protein is involved in aldosterone's natriferic action.