bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of the rat P2u receptor and its potential role in coronary vasodilation
Authors: Godecke S. , Decking U. K. , Godecke A. , Schrader J.
Published: December 2017

We cloned and sequenced the cDNA as well as the genomic DNA of the P2u receptor gene from the rat. The coding region of the gene is not interrupted by introns. P2u is expressed in a variety of rat organs with pronounced differences of expression intensities. Highest expression was found in liver and testis, while no expression could be detected in the brain. High P2u expression was found in primary microvascular endothelial cells from the rat heart, but not in cardiac myocytes. By in situ analysis, we localized P2u expression in epithelial cells of esophagus and bronchi. Functional analysis revealed that, in isolated perfused rat hearts, the P2u ligands UTP and ATP induce a pronounced vasodilation of coronary blood vessels. In contrast, UMP and uridine, the degradative products of UTP, act as potent vasoconstrictors. Our experiments suggest that, in the rat heart, endothelial P2u receptors are involved in the ATP/UTP-mediated vasodilation of coronary blood vessels.
Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry
Authors: Rokaw M. D. , Sarac E. , Lechman E. , West M. , Angeski J. , Johnson J. P. , Zeidel M. L.
Published: December 2017

In several settings in vivo, prolonged inhibition of apical Na+ entry reduces and prolonged stimulation of apical entry enhances the ability of renal epithelial cells to reabsorb Na+, an important feature of the load-dependent regulation of renal tubular Na+ transport. To model this load dependency, apical Na+ entry was inhibited or stimulated for 18 h in A6 cells and vectorial transport was measured as short-circuit current (Isc) across monolayers on filter-bottom structures. Basal amiloride-sensitive Isc represents the activity of apical Na+ channels, whereas Isc after permeabilization of the apical membrane to cations with nystatin represents maximal activity of the basolateral Na(+)-K(+)-ATPase. Chronic inhibition of apical Na+ entry by 18-h apical exposure to amiloride or replacement of apical Na+ with tetramethylammonium (TMA+), followed by washing and restoration of normal apical medium, revealed a persistent decrease in Isc that remained despite exposure to nystatin. Both basal and nystatin-stimulated Isc recovered progressively after restoration of normal apical medium. In contrast, chronic stimulation of apical Na+ entry by short circuiting the epithelium increased Isc in the absence and presence of nystatin, indicating upregulation of both apical Na+ channels and basolateral Na(+)-K(+)-ATPase. Basolateral equilibrium [3H]ouabain binding was reduced to 67 +/- 5% in TMA+ vs. control cells, whereas values in 18-h short-circuited cells increased by 42 +/- 19%. The results demonstrate that load dependency of tubular Na+ transport can be modeled in vitro and indicate that the regulation of Na(+)-K(+)-ATPase observed in these studies occurs in part by changes in the density of functional transporter proteins within the basolateral membrane.
1,2-Dioctanoyl-sn-glycerol depresses cardiac L-type Ca2+ current: independent of protein kinase C activation
Authors: Schreur K. D. , Liu S.
Published: December 2017

The present study examines the effect of 1,2-dioctanoyl-sn-glycerol (DiC8), a diacylglycerol analogue, on L-type Ca2+ current (ICa,L) in adult rat ventricular myocytes using whole cell patch-clamp techniques. Extracellular application of DiC8 (1-10 microM) resulted in a concentration-dependent inhibition of peak ICa,L (half-maximum inhibitory concentration = 2.2 microM). Results obtained from the current-voltage relationship showed that DiC8 decreased the slope conductance. In addition, DiC8 increased the rate of Ba2+ current inactivation and caused a hyperpolarizing shift in the steady-state inactivation by 6 mV and a decrease in the slope factor. The DiC8-induced inhibition of ICa,L was neither mimicked by activation of protein kinase C (PKC) with 100 nM phorbol 12-myristate 13-acetate (PMA) no prevented by inhibition of PKC with 30 microM H-7, 100 nM staurosporine, or 24-h pretreatment with PMA. These results suggest that in rat ventricular myocytes 1) 1,2-sn-diacylglycerol (DAG) inhibits ICa,L, possibly by facilitating channel inactivation and decreasing channel availability and 2) this inhibitory effect of DAG is independent of PKC activation.
Reconstitution of calyculin-inhibited K-Cl cotransport in dog erythrocyte ghosts by exogenous PP-1
Authors: Krarup T. , Dunham P. B.
Published: December 2017

Osmotic swelling of dog and other mammalian erythrocytes activates Cl-dependent K transport, K-Cl cotransport. This activation can be abolished by nanomolar concentrations of calyculin, a potent inhibitor of serine-threonine protein phosphatases. Therefore, K-Cl cotransport is probably activated by dephosphorylation by a type 1 and/or type 2A protein phosphatase (PP-1 and PP-2A, respectively). This was tested directly by incorporating exogenous protein phosphatases into resealed ghosts made from dog erythrocytes previously exposed to calyculin. K-Cl cotransport was nearly completely inhibited in the ghosts. Incorporation of PP-1 reconstituted K-Cl cotransport. Maximal reconstitution was up to 90% of the control flux in the ghosts and 0.1 U PP-1/ml lysate gave half-maximal reconstitution of cotransport. In contrast, PP-2A had no effect. This result with PP-1 provides direct evidence that K-Cl cotransport is activated by PP-1 in dog erythrocytes. Half-maximal activation of K-Cl cotransport required approximately 180 molecules of PP-1 per ghost.
Effect of purinergic blockers on outward current in isolated smooth muscle cells of the sheep bladder
Authors: Cotton K. D. , Hollywood M. A. , Thornbury K. D. , McHale N. G.
Published: December 2017

Freshly dispersed cells from sheep urinary bladder were voltage clamped using the whole cell and inside-out patch-clamp technique. Cibacron and Basilen blue increased outward current in a dose-dependent manner with a half-maximal response at 10(-5)M. Suramin, in concentrations to 10(-3)M, had no such effect. The Cibacron blue response was abolished in Ca2+ -free physiological salt solution, suggesting that it was acting on a Ca2+ -dependent current. Similarly, the Cibacron blue-sensitive current was significantly attenuated by charybdotoxin. Cibacron blue did not modulate inward current nor were its effects modified by caffeine or heparin, suggesting that its effect on outward current was not secondary to an increase in intracellular Ca2+. Application of 10(-4)M Cibacron blue to the inside membrane of excised patches caused a rapid increase in open probability of a large conductance (300 pS) K+ channel. These results suggest that Cibacron blue is a potent activator of a Ca2+ -dependent outward current in bladder smooth muscle cells in addition to its action as a purinergic blocker.
Stable expression of a human HSP70 gene in a rat myogenic cell line confers protection against endotoxin
Authors: Chi S. H. , Mestril R.
Published: December 2017

Recent reports show that a pre-heat shock has a protective effect against endotoxin “in vivo” in rodents. It has remains unclear what actually confers the protection against endotoxin. One candidate for this protective effect is the heat shock protein of 70 kDa (HSP70). We found that a mild heat shock pretreatment is the rat myogenic cell line, H9c2(2-1), confers resistance to a subsequent exposure to endotoxin. A myogenic rat cell line stably transfected with the human inducible HSP70 exhibits an increased survival rate compared with cells stably transfected solely with the selectable neomycin marker gene or the parental cell line H9c2(2-1) when exposed to endotoxin. The mechanism of endotoxin-induced cell injury is postulated to be through the generation of nitric oxide in these myogenic cells during exposure to endotoxin. We conclude that HSP70, regardless of the particular mechanism of cytotoxicity, plays a role in protecting the cell against the deleterious effects of endotoxin.
Regulation of taurine transport in rat astrocytes by protein kinase C: role of calcium and calmodulin
Authors: Tchoumkeu-Nzouessa G. C. , Rebel G.
Published: December 2017

Phorbol 12-myristate 13-acetate, a potential stimulator of protein kinase C (PKC), inhibited taurine uptake in rat astrocytes. This effect was mimicked by 1-oleoyl-2-acetyl-sn-glycerol, an endogenous stimulator of PKC, and by r-59949, an inhibitor of diacylglycerol kinase. Maximal inhibition was obtained at microM phorbol 12-myristate 13-acetate (PMA) after 1 h of treatment. This effect was prevented by pretreatment of the cells with chelerythrine, a potent and selective inhibitor of PKC. The transport of beta-alanine, an amino acid that shares the same transporter as taurine, was inhibited to a comparable extent. The effect of PMA was potentiated by cotreatment of the cells with thapsigargin or the Ca2+ ionophore A-23187. However, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N1,N1-tetraacetic acid and verapamil did not prevent the PMA effect. Pretreatment of the cells with calmodulin antagonists W-13 or calmidazolium, prevented the PMA-induced inhibition of taurine uptake. This inhibition was not affected by cycloheximide, actinomycin D, colchicine, or cytochalasin D. The Na(+)-to-Cl(-)-to-taurine coupling ratio was unaffected. Dimethyl amiloride, a selective inhibitor of Na+/H+ antiport, was unable to prevent the effects of PMA. These effects were associated with a decrease in the maximal velocity and an increase in the Michaelis-Menten constant.
Mechanism of swelling activation of K-Cl cotransport in inside-out vesicles of LK sheep erythrocyte membranes
Authors: Kelley S. J. , Dunham P. B.
Published: December 2017

Stimulation by swelling of K-Cl cotransport was studied in inside-out vesicles (IOVs) made from membranes of LK sheep erythrocytes. The purpose was to understand this stimulation in terms of the three-state process proposed for regulation of the cotransporter (P.B. Dunham, J. Klimczak, and P.J. Logue. J. Gen. Physiol. 101: 733-765, 1993). The first step in this process, A --> B, is rate limiting and controlled by transphosphorylation reactions. The second step, B --> C, is fast; its control is unknown. Predictions were that maximum velocity (Jmax) of cotransport increases with A --> B and concentration at one-half Jmax (K1/2) of K+ as a substrate decreases with B --> C. We tested the hypothesis that most transporters in IOVs are in the B state and that swelling activates cotransport in vesicles by the B --> C conversion. In accordance with this hypothesis, swelling should activate K+ influx with no discernable delay. It did. K1/2 for K+ should decrease with swelling and Jmax should not change. K1/2 decreased 10-fold, and Jmax did not change. Inhibitors of transphosphorylation, reactions of A --> B, should not affect K+ flux into IOVs, and they did not. The results support the hypothesis: swelling activation of K+ flux into IOVs corresponds to B --> C. A mechanical change in the membrane causes a specific change in the cotransporter: an increase in apparent affinity for K+.
Contraction-independent effects of catecholamines on glucose transport in isolated rat cardiomyocytes
Authors: Fischer Y. , Thomas J. , Holman G. D. , Rose H. , Kammermeier H.
Published: December 2017

The effects of catecholamines on glucose transport were studied in noncontracting isolated rat cardiomyocytes. alpha-Adrenergic treatment (phenylephrine, or norepinephrine + propranolol) led to an approximately fourfold stimulation of glucose transport in basal cells (no insulin). The effect of phenylephrine was suppressed by the alpha 2-antagonist yohimbine or the beta-antagonist propranolol. The beta-adrenergic agonist isoproterenol partially counteracted the action of phenylephrine (but not that of insulin). Phenylephrine increased glucose transport in two phases with apparent half times of 3.2 and 13.0 min, respectively. Correspondingly, different EC50 values were found after 10 and 45 min on phenylephrine addition (5.0 +/- 1.9 vs. 31.6 +/- 9.6 microM, respectively). Maximal stimulation by phenylephrine was at least partially additive to that of insulin and of other stimulators of glucose transport (e.g., H2O2, vanadate, lithium). Phenylephrine significantly increased the level of cell surface glucose carriers GLUT-1 (1.54-fold) and GLUT-4 (1.78-fold), as assessed by using the specific photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]- 1,3-bis(D-mannos-4-yloxy)propyl-2-amine. In conclusion, catecholamines stimulate cardiomyocyte glucose transport through alpha 1-adrenergic receptors independently or downstream of a contraction-evoked stimulus. This effect is at least partially explained by a recruitment of glucose transporters to the cell surface. The mechanism(s) and/or signals involved differ from those triggered by insulin and insulinomimetic agents.
Whole cell and unitary amiloride-sensitive sodium currents in M-1 mouse cortical collecting duct cells
Authors: Chalfant M. L. , O'Brien T. G. , Civan M. M.
Published: December 2017

Amiloride-sensitive whole cell currents have been reported in M-1 mouse cortical collecting duct cells (Korbmacher et al., J. Gen. Physiol. 102: 761-793, 1993). We have confirmed that amiloride inhibits the whole cell currents but not necessarily the measured whole cell currents. Anomalous responses were eliminated by removing external Na+ and/or introducing paraepithelial shunts. The amiloride-sensitive whole cell currents displayed Goldman rectification. The ionic selectivity sequence of the amiloride-sensitive conductance was Li+ > Na+ >> K+. Growth of M-1 cells on permeable supports increased the amiloride-sensitive whole cell permeability, compared with cells grown on plastic. Single amiloride-sensitive channels were observed, which conformed to the highly selective low-conductance amiloride-sensitive class [Na(5)] of epithelial Na+ channels. Hypotonic pretreatment markedly slowed run-down of channel activity. The gating of the M-1 Na+ channel in excised patches was complex. Open- and closed-state dwell-time distributions from patches that display one operative channel were best described with two or more exponential terms each. We conclude that 1) study of M-1 whole cell Na+ currents is facilitated by reducing the transepithelial potential to zero, 2) these M-1 currents reflect the operation of Na(5) channels, and 3) the Na+ channels display complex kinetics, involving > or = 2 open and > or = 2 closed states.
In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts
Authors: Zhang Y. , Yankaskas J. , Wilson J. , Engelhardt J. F.
Published: December 2017

An in vivo human bronchial xenograft model system was used to simultaneously analyze electrolyte and fluid transport defects in fully differentiated human cystic fibrosis (CF) and non-CF proximal airways. CF airways demonstrated three discernible defects when compared with non-CF, including 1) a lack of adenosine 3',5'-cylic monophosphate (cAMP)-inducible Cl- secretion, 2) a fourfold higher basal fluid absorption rate, and 3) an altered regulation of fluid absorption in response to amiloride-stimulated changes in Na+ transport. A unique finding in this study demonstrated that treatment of epithelia with amiloride led to a greater than threefold decrease in the rate of fluid absorption in CF tissues as contrasted to a greater than threefold increase in the rate of fluid absorption in non-CF tissues. The removal of apical Na+ from amiloride-treated non-CF xenografts was capable of ablating this amiloride-induced increase in fluid absorption. In light of the recent interactions demonstrated between CF transmembrane conductance regulator (CFTR) and the rat epithelial, amiloride-sensitive Na+ channel, these findings implicate additional complexities between the Na+ conductance pathways and fluid transport in normal and CF proximal airways. Such findings suggest that CFTR may also regulate amiloride-insensitive Na+ channels.
P-glycoprotein-associated chloride currents revealed by specific block by an anti-P-glycoprotein antibody
Authors: Han E. S. , Vanoye C. G. , Altenberg G. A. , Reuss L.
Published: December 2017

The relationships between P-glycoprotein (PGP) expression and plasma membrane ion currents activated by cell swelling were studied in several cell lines by use of the whole cell configuration of the patch-clamp technique. Swelling-activated Cl- currents (ICls) had similar characteristics independently of whether PGP was expressed. Addition of the anti-PGP monoclonal antibody C219 or its Fab fragment to the pipette solution prevented ICls in cells expressing functional PGP (assessed by immunoblots, immunofluorescence, and transport of rhodamine 123) but not in cells lacking PGP expression. A peptide analogue of the C219 epitope abolished the effect of C219. Other anti-PGP antibodies and mouse immunoglobulin G were ineffective. C219 did not alter swelling-activated cation currents. Inasmuch as ICls is present in cells that do not express PGP and C219 has no effect on ICls in these cells, we conclude that PGP is not required for the ICls phenotype. However, when expressed in the plasma membrane, PGP is involved, directly or indirectly, in ICls but not in swelling-activated K+ currents.
Tyrosine phosphorylation and activation of pp60c-src and pp125FAK in bradykinin-stimulated fibroblasts
Authors: Lee K. M. , Villereal M. L.
Published: December 2017

Bradykinin (BK) stimulates protein tyrosine phosphorylation in human foreskin fibroblasts (K.-M. Lee, K. Toscas, and M. L. Villereal, J. Biol. Chem. 268:9945-9948, 1993). The major tyrosine phosphorylation occurs in proteins of a molecular mass of 130 and 70 kDa. In this report, we demonstrate that focal adhesion-associated tyrosine kinase, pp125FAK, is one component of the 130-kDa phosphotyrosine band. The BK-stimulated pp125FAK tyrosine phosphorylation level is well correlated with increased kinase activity, as assessed by in vitro immune complex kinase assays. We have identified paxillin, a protein that is localized in focal adhesions, as a component of the 70-kDa phosphotyrosine band. In addition to identifying the two proteins responsible for the major phosphotyrosine bands, we also report that pp60c-src is tyrosine phosphorylated and activated in response to BK, as analyzed by immunoblotting and in vitro kinase assays, respectively. These findings indicate, for the first time, that the BK receptor is coupled to the important protooncogene c-src and that the src pathway may mediate some of the events downstream from BK binding.
Effects of hypercapnia on prostanoid and cAMP production by cerebral microvascular cell cultures
Authors: Parfenova H. , Leffler C. W.
Published: December 2017

In the newborn pig cerebral circulation, arteriolar dilation in response to hypercapnia requires the presence of intact endothelium and is accompanied by an indomethacin-sensitive increase in cortical adenosine 3',5'-cyclic monophosphate (cAMP). The effects of short-term hypercapnia on production of dilator prostanoids and cAMP were investigated using newborn pig cerebral microvascular smooth muscle cells and endothelial cells cultured both separately and in noncontact coculture. Microvascular smooth muscle cells respond to hypercapnia (pH 7.00 +/- 0.05 PCO2 75 +/- 3 mmHg) by a 1.3- to 1.7-fold increase in basal cAMP production that is not affected by indomethacin, whereas hypercapnia and 80 mM sodium propionate do not affect iloprost-stimulated cAMP production. Microvascular endothelial cells cultured on Millicel inserts respond to hypercapnia by a two- to fourfold increase in prostacyclin (as 6-keto-prostaglandin F1 alpha) and prostaglandin E2 production in both luminal and abluminal compartments. For noncontact coculture, Millicel inserts with endothelial cells (as hypercapnia-sensitive producers of prostanoids) were installed into cell culture dishes with aspirin-pretreated smooth muscle cells (as targets for endothelium-derived dilator prostanoids). Exposure of noncontact microvascular cell cocultures to hypercapnia results in a three- to fourfold stimulation of prostanoid and cAMP production. Therefore, short-term hypercapnia increases cAMP production by microvascular smooth muscle cells via 1) a direct (prostanoid independent) mechanism and 2) an endothelial-dependent pathway that involves prostanoids. Endothelium-produced prostanoid signals are necessary for a full increase in cAMP production by cerebral microvascular smooth muscle cells in response to hypercapnia.
Cardiac sodium channels expressed in a peripheral neurotumor-derived cell line, RT4-B8
Authors: Zeng D. , Kyle J. W. , Martin R. L. , Ambler K. S. , Hanck D. A.
Published: December 2017

RT4-B is one of several cell lines derived from a multipotent stem cell line, RT4-AC, which originated from a rat peripheral neurotumor. Based on Northern blot and ribonuclease protection experiments, RT4-B8 cells have been proposed to express rat cardiac Na channel mRNA as the major isoform. We report here direct electrophysiological evidence that the expressed voltage-gated Na channels in the RT4-B8 cell line are of the cardiac phenotype with no evidence for subpopulations expressing other Na channel isoforms. Current activation half point (conductance) was -41 +/- 5 mV (n = 7) and the steady-state voltage-dependent availability half point was -89 +/- 1 mV. As expected for cardiac Na channels, the half concentration of block for tetrodotoxin block was 0.74 microM, for saxitoxin (STX) was 0.15 microM, and for the class 2B divalent cation Cd2+ was 67 microM. Block was well described by single-site dose-response relationships with no indication of a subpopulation with "neuronal" affinity. Single-channel conductance (140 mM Na+) was 10 pS and predicted the average number of channels open at peak Na current to be 3 channels/microns2. [3H]STX binding data were also consistent with a single population of low-affinity STX binding sites and predicted channel density to be 11 sites/microns2. No inwardly or outwardly rectifying K or Ca currents were detected electrophysiologically, although in some cells a small time-independent Cl current was detected. Reverse transcription-polymerase chain reaction of mRNA isolated from RT4-B8 cells demonstrated the presence of rat cardiac (rH1) and brain IIa alpha-subunit mRNA, as well as mRNA for the Na channel beta 1-subunit. Northern blot analysis confirmed the predominance of the rat cardiac Na mRNA compared with brain IIa. The beta 1-subunit mRNA levels were significantly lower than those detected in rat brain and heart mRNA but were comparable to the low level of beta 1-subunit mRNA detected in isolated rat ventricular myocytes.
cAMP-independent regulation of CFTR by the actin cytoskeleton
Authors: Prat A. G. , Xiao Y. -F. , Ausiello D. A. , Cantiello H. F.
Published: December 2017

Pages C1552-C1561: A. G. Prat, Y.-F. Xiao, D. A. Ausiello, and H. F. Cantiello. “AMP-independent regulation of CFTR by the actin cytoskeleton.” Page C1558, Fig. 8A: units on the y-axis of the bar graph should have been 10 times larger. The corrected figure appears below.

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Nucleotide receptor-mediated decrease of tight-junctional permeability in cultured human cervical epithelium
Authors: Gorodeski G. I. , Peterson D. E. , De Santis B. J. , Hopfer U.
Published: December 2017

Extracellular ATP changes the transepithelial electrical conductance (GT) across cultures of human cervical cells acutely, in a biphasic manner that is characterized by a rapid increase (phase I) followed by a sustained decrease in GT (phase II). We tested the hypothesis that the phase II response is mediated by decreases in the permeability of tight junctions. We studied the effect of ATP on the relative mobilities of Cl- vs. Na+ (uCl/uNa) as calculated from changes in the dilution potential (Vdil). Vdil was induced by lowering NaCl from 130 to 10 mM in either the luminal or subluminal solutions bathing filters containing cells. uCl/uNa was 1.27 across cervical cultures and 1.34 across blank filters, compared with a level of 1.52 in free solution. Increases in GT induced by transepithelial hydrostatic or hypertonic gradients (which increase permeability of lateral intercellular space) had no effect on uCl/uNa. Increases in GT induced by lowering extracellular Ca2+ to < 0.1 mM increased uCl/uNa to levels obtained in blank filters, indicating abrogation of tight-junctional resistance. Phase I response and ionomycin (which produces a sustained phase I-like increase in GT) had no effect on uCl/uNa. The phase II response, however, decreased uCl/uNa from 1.27 to 1.24, and the effect could be abrogated by lowering extracellular Ca2+. These results indicate that phase II decreases in GT across cultured human cervical epithelium are mediated by acute decreases in tight-junctional permeability.
Allosteric modulation of GABAA receptors in acutely dissociated neurons of the suprachiasmatic nucleus
Authors: Shimura M. , Harata N. , Tamai M. , Akaike N.
Published: December 2017

The gamma-aminobutyric acid (GABA)-induced response was investigated in acutely dissociated suprachiasmatic nucleus (SCN) neurons of 11- to 14-day-old rats, under the voltage-clamp condition of nystatin-perforated patch recording. At a holding potential of -40 mV, application of GABA induced inward currents in a concentration-dependent manner. Pentobarbital and 5 beta-pregnan-3 alpha-ol-20-one (pregnanolone) similarly induced inward currents. GABA-induced inward currents were suppressed in a concentration-dependent manner by pretreating neurons with a GABAA receptor antagonist, bicuculline. Bicuculline (3 x 10(-6) M) shifted the concentration-response curve of GABA to the left in a competitive manner. Reversal potential of the GABA response (EGABA) was -3.4 +/- 0.7 mV, close to the theoretical Cl- equilibrium potential of -4.1 mV. Pretreating SCN neurons with diazepam, pentobarbital, and pregnanolone enhanced the 3 x 10(-6) M GABA response. Diazepam (3 x 10(-8) M), pentobarbital (3 x 10(-5) M), and pregnanolone (10(-7) M) shifted the concentration-response curve of GABA to the left without changing the maximal amplitude of GABA responses. EGABA in the presence of diazepam, pentobarbital, or pregnanolone was the same as that in their absence. These results show that the GABA response in acutely dissociated SCN neurons is mediated by the GABAA receptor. Because the GABAA receptor of SCN neurons is allosterically augmented by diazepam, pentobarbital, and pregnanolone, similarly as in other regions of the central nervous system, the present study opens up ways to functionally modulate the GABAA receptors in SCN.
Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels
Authors: Chen Y. , Simasko S. M. , Niggel J. , Sigurdson W. J. , Sachs F.
Published: December 2017

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.
Endothelin and nitric oxide release modulate aortic contraction to selected thrombin receptor agonists
Authors: Magazine H. I. , Butt O. , Yaghoutiel H. R.
Published: December 2017

The contribution of endothelin-1 (ET-1) and nitric oxide (NO) release to vascular contraction induced by synthetic peptide agonists of the alpha-thrombin receptor, TRAP-14 and TRAP-6, was evaluated with the use of rings of rat aorta. TRAP-6 induced fourfold greater contraction than that induced by addition of TRAP-14. TRAP-14, but not TRAP-6, stimulation of aortic rings resulted in a rapid (in seconds) and dose-dependent increase in ET-1 levels detected in the vessel perfusate. Release of ET-1 in vessels denuded of endothelium was indistinguishable from that of intact vessels, suggesting that endothelial cells are not required for the observed ET-1 release. The contractile potency of TRAP-14 was reduced in the presence of BQ-123, a specific antagonist of the endothelin A subtype of ET receptors, whereas TRAP-14 potency was increased significantly by pretreatment with the NO synthetase inhibitor NG-nitro-L-arginine (L-NNA). The contractile potency of TRAP-6 was not altered in the presence of BQ-123 or L-NNA, suggesting that TRAP-14 but not TRAP-6 potency is modulated by ET-1 and NO release. These data indicate that TRAP-6 has limited function relative to TRAP-14 and that thrombin receptor activation is not sufficient to induce ET-1 and NO release from rat aorta.
A role for CFTR in human autosomal dominant polycystic kidney disease
Authors: Hanaoka K. , Devuyst O. , Schwiebert E. M. , Wilson P. D. , Guggino W. B.
Published: December 2017

Pages C389-C399: K. Hanaoka, O. Devuyst, E. M. Schwiebert, P. D. Wilson, and W. B. Guggino. “A role for CFTR in human autosomal dominant polycystic kidney disease.” Page C395, Fig. 5: because of a photographic processing error, panels B and D, which both show unstained cells, were mislabeled and incorrectly reproduced. The corrected figure appears below.

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Ca2+- and pH-dependent halothane stimulation of Ca2+ release in sarcoplasmic reticulum from frog muscle
Authors: Beltrán M. , Bull R. , Donoso P. , Hidalgo C.
Published: December 2017

Pages C540-C546: M. Beltrán, R. Bull, P. Donoso, and C. Hidalgo. “Ca2+- and pH-dependent halothane stimulation of Ca2+ release in sarcoplasmic reticulum from frog muscle.” Page C543, because of a printer's error, Fig. 6 was inadvertently published as a repeat of Fig. 5. The correct Fig. 6 appears below.

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Dipeptide uptake by adenohypophysial folliculostellate cells
Authors: Otto C. , tom Dieck S. , Bauer K.
Published: December 2017

Dipeptide uptake was studied in primary cultures from rat anterior pituitaries by use of radiolabeled carnosine and the fluorescent dipeptide derivative beta-Ala-Lys-N epsilon-AMCA (AMCA is 7-amino-4-methylcoumarin-3-acetic acid). Fluorescence microscopic studies revealed that the reporter peptide specifically accumulated in the S-100 positive folliculostellate cells that do not produce any known hormone. The dipeptide derivative was taken up in unmetabolized form by an energy-dependent saturable process with apparent kinetic constants as follows: Michaelis constant, 19 microM; maximum velocity, 5.5 nmol.mg protein-1.h-1. This high-affinity transporter was strongly affected by inhibitors of sodium/proton exchangers and thus appeared to be driven by a proton gradient. Competition studies revealed that the peptide transporter exhibits broad substrate specificity with a preference for hydrophobic dipeptides. In contrast to free amino acids and the pseudotetrapeptide amastatin, tripeptides were also accepted. Compounds without an alpha- and beta-amino group, such as captopril, thiorphan, and benzylpenicillin, did not affect uptake of the reporter peptide, although they were substrates of the well-characterized intestinal and renal dipeptide transporters.
Interactions of external and internal K+ with K(+)-HCO3- cotransporter of rat medullary thick ascending limb
Authors: Blanchard A. , leviel F. , Bichara M. , Podevin R. A. , Paillard M.
Published: December 2017

We studied [K+]i and [K+]o, where subscripts i and o refer to intracellular and extracellular, respectively, concentration dependency of the kinetic properties of the electroneutral K(+)-HCO3-cotransport, using suspensions of rat medullary thick ascending limb (mTAL). With the use of nigericin and monensin, [K+]i was clamped at various values, while maintaining [Na+]i = [Na+]o = 37 mM, [HCO3-]i = [HCO3-]o = 23 mM, and pHi = pHo = 7.4. As indicated by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein HCO3(-)-dependent rates of change in pHi, at constant [K+]i, increasing the magnitude of the outward K+ gradient by varying [K+]o saturated HCO3-efflux with a Michaelis-Menten curve (apparent Michaelis constant for [K+]o = 2 mM, Hill coefficient = 1). On the other hand, increasing [K+]i from 30 to 140 mM, while either [K+]o or the magnitude of the K+ concentration gradient was fixed, saturated HCO3- efflux with a sigmoidal curve and yielded a Hill coefficient of 3.4 and 50% of maximum velocity at 70 mM [K+]i. These results indicate that [K+]i, independent of its role as a transportable substrate for the cotransport with HCO3-, has a role as an allosteric activator of the K(+)-HCO3- cotransporter. Such an allosteric modulation may contribute to the maintenance of net HCO3- absorption despite large in vivo physiological variations of K+ concentration in the medullary interstitium.
Apical membrane permeability of MDCK cells
Authors: Rivers R. L. , McAteer J. A. , Clendenon J. L. , Connors B. A. , Evan A. P. , Williams J. C.
Published: December 2017

The osmotic water permeability (Pf) and permeability to nonelectrolytes were determined for the apical membrane of clonal strain Madin-Darby canine kidney (MDCK) C12 cells cultured as cysts with the apical membrane facing the surrounding medium. Pf and solute permeabilities were calculated from the rate of volume change of cysts by digitizing images at 1-s intervals after instantaneous osmotic challenge. Image measurement was fully automated with the use of a program that separated the image of the cyst from the background by using adaptive intensity thresholding and shape analysis. Pf, calculated by curve fitting to the volume loss data, averaged 2.4 +/- 0.1 micron/s and was increased by addition of amphotericin B. The energy of activation for Pf was high (16.3 kcal/mol), and forskolin (50 microM) had no effect on Pf. Two populations of MDCK cysts were studied: those with two to three cells and those that appeared to be composed of only one cell. The Pf of multicell cysts was the same as single cell cysts, suggesting that paracellular water flow is not significant. Solute permeability was measured using paired osmotic challenges (sucrose and test solute) on the same cyst. Urea permeability was not different from zero, whereas the permeabilities of acetamide and formamide were consistent with their relative oil-water partition coefficients. Our data are similar to values from studies on the permeability properties of vesicles of water-tight epithelial apical membrane. The combination of the unique model of MDCK apical-out cysts and fully automated data analysis enabled determination of apical membrane permeability in intact epithelial cells with high precision.
Inhibitory effect of Ca2+ on renin secretion elicited by chemiosmotic stimuli through actomyosin mediation
Authors: Park C. S. , Lee H. S. , Chang S. H. , Honeyman T. W. , Hong C. D.
Published: December 2017

We had previously shown that several experimental manipulations, which are likely to produce osmotic swelling of renin secretory granules, stimulate secretion of renin (C.S. Park, T.W. Honeyman, S. K. Ha, H. K. Choi, C. L. Chung, and C. D. Hong. J. Pharmacol. Exp. Ther. 259: 211-218, 1991). In subsequent studies, Ca2+ was found to block the stimulation of renin secretion evoked by osmotic swelling of renin secretion granules [Park, Hong, and Honeyman, Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F793-F798, 1992]. Furthermore, evidence from our recent studies indicates that myosin light chain kinase (MLCK) might be involved in the inhibition of renin secretion through Ca(2+)-calmodulin. In the present study we investigate the possibility that MLCK might mediate the inhibitory action of Ca2+ on renin secretion stimulated by osmotic swelling of renin secretory granules. Rat renal cortical slices were incubated under a variety of experimental conditions that would produce osmotic swelling of renin secretory granules. Incubation in hypotonic KCl medium, isosmotic NH4Cl or CH3COONH4 medium, or isosmotic KCl or CH3COOK medium plus nigericin in the absence of Ca2+ all produced a significant increase in renin secretion 2- to 14-fold (P < 0.001). Ca2+ added to all of these media partially or completely blocked the stimulatory effects (P < 0.001). This inhibitory effect of Ca2+ was significantly blocked by ML-9 (10(-4) M, P < 0.001), a putative specific inhibitor of the Ca(2+)-calmodulin-dependent MLCK. Taken together, the present findings support the idea that the renin secretory response may involve chemiosmotic swelling of renin secretory granules. This pivotal step may be regulated by contractile actomyosin interaction, which is in turn modulated through the Ca(2+)-calmodulin-dependent activity of MLCK.
Action of protein kinase C in endothelin-induced contractions in rat aortic rings
Authors: Oriji G. K. , Keiser H. R.
Published: December 2017

Endothelin (ET) is a potent vasoconstrictor peptide that induces characteristically long-lasting contractions. We used both intact and endothelium-denuded rat aortic rings to investigate the role of protein kinase C (PKC) in ET-induced contractions. ET (10(-9) M) and phorbol 12,13-dibutyrate (PDBu), a PKC activator, produced a gradual and sustained contraction of greater magnitude in denuded aortic rings than in intact rings. When aortic rings were pretreated with graded concentrations of different PKC inhibitors, inhibition of ET-induced contractions began at 10(-9)M and was nearly complete at 10(-3)M, and the reduction was greater in intact than in denuded rings. Pretreatment of aortic rings with PDBu or NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, potentiated ET-induced contractions. PKC enzyme assay showed activation of PKC in aortic rings that were treated with either ET or PDBu, inhibition after pretreatment with PKC inhibitors, and no change with 4 alpha-phorbol 12,13-didecanoate (PDD), an inactive phorbol ester. ET significantly increased nitrate and nitrite production, which was further increased by pretreatment with PKC inhibitors. PDBu prevented ET-induced nitrate/nitrite production, and PDD had no effect. These results strongly suggest that PKC mediates, in part, ET-induced contractions in rat aortic rings and that an intact endothelium is required for maximum inhibition by PKC inhibitors because PKC stimulated by ET inhibits nitric oxide release.
Protein kinase A activation is required for IL-1-induced nitric oxide production by cardiac myocytes
Authors: Oddis C. V. , Simmons R. L. , Hattler B. G. , Finkel M. S.
Published: December 2017

We have previously reported that interleukin-1 beta (IL-1) alone induced the transcription of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production by isolated neonatal rat cardiac myocytes (CM). The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. The addition of IL-1 to CM resulted in a peak rise in both adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PKA) activities by 10 min followed by rapid declines and return to basal levels within 60 min. The PKA inhibitor KT-5720 completely blocked NO-2 production by IL-1-stimulated CM (P < 0.01; n = 12). The protein kinase C (PKC) inhibitor, calphostin C, had no effect on NO2- production by IL-1 stimulated CM [P = not significant (NS); n = 12]. The addition of PKA+cAMP to cytosols derived from IL-1-treated CM did not directly enhance iNOS enzyme activity (P = NS; n = 3). CM treated with IL-1 alone stained positively for iNOS protein by immunohistochemistry. iNOS staining was absent in CM treated with IL-1+KT-5720. KT-5720 resulted in an earlier disappearance of iNOS mRNA from IL-1-treated CM, as detected by semiquantitative reverse transcriptase-polymerase chain reaction. We report for the first time that PKA (but not PKC) activation is required for IL-1-induced NO production by CM.
Signal transduction and glial cell modulation of cultured brain microvessel endothelial cell tight junctions
Authors: Raub T. J.
Published: December 2017

Noncontact coculture of postconfluent bovine brain microvessel endothelial cell (BMEC) monolayers with rat C6 glioma cells results in markedly decreased transmonolayer permeability measured by transendothelial electrical resistance (TER) and solute flux. An elevation in adenosine 3',5'-cyclic monophosphate (cAMP) in response to forskolin correlates with an increase in TER through a threshold event; however, unlike forskolin, the severalfold increase in TER induced by C6 cells is cAMP independent. Activation of protein kinase C (PKC) enhances the C6 cell-induced increase in TER, and PKC inhibition blocks C6 cell induction. Treatment of control or C6 cell-induced BMEC monolayers with pertussis toxin immediately and irreversibly obliterates TER, without an apparent change in guanosine 3',5'-cyclic monophosphate levels or viability, indicating the importance of a G protein-mediated event. A similar effect is observed with transforming growth factor-beta 1, and both responses are polarized, since the loss in TER is significantly faster in BMEC monolayers exposed basolaterally. These results suggest that astroglia modulate the blood-brain barrier through a cAMP-independent PKC-dependent pathway maintained by a G protein-coupled mechanism.
Anion channel blockers inhibit swelling-activated anion, cation, and nonelectrolyte transport in HeLa cells
Authors: Hall J. A. , Kirk J. , Potts J. R. , Rae C. , Kirk K.
Published: December 2017

The effect of osmotic cell swelling on the permeability of HeLa cells to a range of structurally unrelated solutes including taurine, sorbitol, thymidine, choline, and K+ (96Rb+) was investigated. For each solute tested, reduction in the osmolality of the medium from 300 to 200 mosmol/kgH2O caused a significant increase in the unidirectional influx rate. In each case, the osmotically activated transport component was nonsaturable up to external substrate concentrations of 50 mM. Inhibitors of the swelling-activated anion channel of HeLa cells [quinine, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, niflumate, 1,9-dideoxyforskolin, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and tamoxifen] blocked the osmotically activated influx of each of the different substrates tested, as well as the osmotically activated efflux of taurine and I-. Tamoxifen and NPPB were similarly effective at blocking the osmotically activated efflux of 96Rb+. The simplest of several hypotheses consistent with the data is that the osmotically activated transport of the different solutes tested here is via a swelling-activated anion-selective channel that has a significant cation permeability and a minimum pore diameter of 8-9 A.
High permeation of L-type Ca2+ channels at physiological [Ca2+]: homogeneity and dependence on the alpha 1-subunit
Authors: Gollasch M. , Ried C. , Liebold M. , Haller H. , Hofmann F. , Luft F. C.
Published: December 2017

Molecular cloning has identified multiple isoforms of dihydropyridine-sensitive C-class L-type Ca2+ channels. We tested the hypotheses that L-type (C-class) channels exhibit homogeneous high permeation properties at physiological Ca2+ concentrations and membrane potentials. We measured unitary currents through single dihydropyridine-sensitive omega-conotoxin-insensitive endocrine and smooth muscle L-type Ca2+ channels in rat pituitary GH3 and rat aortic A7r5 cell lines. We also measured unitary currents through smooth muscle (Cb) Ca2+ channel alpha 1-subunits in Chinese hamster ovary (CHO) cells. Our results show that single channel conductances of all three L-type (C-class) channels are uniform with high Ba2+ concentrations, e.g., approximately 23 pS with 110 mM Ba2+. The single channel conductances were reduced to similar values when the Ba2+ concentration was lowered to near-physiological values: 11.1, 9.3, and 8.4 pS in GH3, A7r5, and CHO cells at 2 mM Ba2+, respectively. The single channel conductances were not significantly different with near-physiological Ca2+ concentrations: 5.5, 5.9, and 4.9 pS in GH3, A7r5, and CHO cells at 2 mM Ca2+, respectively. The data suggest that L-type (C-class) channels are homogeneous in terms of Ca2+ permeation at physiological charge carrier concentrations and membrane potentials. Furthermore, the data indicate that the relatively high Ca2+ permeation under physiological conditions is determined by the intrinsic properties of the pore-forming Ca2+ channel alpha 1-subunit.
Endothelin-1-evoked arachidonic acid release: a Ca(2+)-dependent pathway
Authors: Wu-Wong J. R. , Dayton B. D. , Opgenorth T. J.
Published: December 2017

Endothelins (ET) are potent vasoconstricting peptides with 21 amino acid residues. Endothelin-1 (ET-1) stimulates arachidonic acid (AA) release in human pericardial smooth muscle cells (HPSMC), which is primarily mediated through the ETA receptor. Manoalide, an inhibitor for phospholipase A2, inhibited the ET-1-evoked response by 50% at 1 microM. RHC-80267, an inhibitor for diacylglycerol lipase, did not have a significant effect. The Ca2+ ionophore A-23187 at 2 microM greatly stimulated AA release in the presence of extracellular Ca2+. ET-1 (10 nM) evoked a robust Ca2+ response. The intracellular Ca2+ concentration reached a peak after 10 s and gradually decreased to a new plateau level in the presence of extracellular Ca2+. ET-1-evoked AA release closely followed the change in the intracellular Ca2+ concentration. Removal of extracellular Ca2+ or treating cells with 250 microM bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM; an intracellular Ca2+ chelator) greatly reduced ET-1-stimulated AA release. The protein kinase C (PKC) inhibitors, staurosporine (1 microM) and chelerythrine chloride (2.5 microM), inhibited ET-1-evoked AA release by 70%. Phorbol 12-myristate 13-acetate, a PKC activator, potentiated the effect of ET on AA release. The data suggest that the effect of ET on AA release in HPSMC is via phospholipase A2, which is modulated by Ca2+ and PKC.
Thrombin-induced vascular reactivity is modulated by ETB receptor-coupled nitric oxide release in rat aorta
Authors: Magazine H. I. , Srivastava K. D.
Published: December 2017

The role of endothelin (ET) receptors in thrombin-induced modulation of vascular tone was evaluated by direct measurement of ET-1 and ET receptor-coupled nitric oxide (NO) release and developed isometric tension in thrombin-treated aortic rings. Here we report that rapid release of ET-1 and subsequent ETB receptor activation are required for production of the potent vasodilator NO by thrombin-stimulated aorta. Thrombin-induced NO release is ablated by pretreatment with ETB receptor antagonists or after ET receptor desensitization by repeated stimulation with ET-1. Thrombin-induced relaxation of precontracted vessels was abrogated in the presence of ETB receptor antagonists and, in contrast, marked contraction to thrombin was observed. These data indicate that the endothelium-dependent vasodilator activity previously attributed to thrombin is indirect and requires ETB receptor-coupled NO release and suggest that ET receptor modulation of thrombin-induced vascular tone may contribute to the increased vasomotor tone observed in diseased and mechanically injured vessels.