Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms.

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC(50)) of <1 n, and had 30-50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL-JAK2 fusion gene; IC(50)=11-120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs.

Cerebral ganglioneuroblastoma in a golden retriever dog.

An 8-month-old Golden Retriever dog was euthanatized because of a large cerebral mass extending from the right frontal lobe to the thalamus that was composed of both mature and immature neuronal cells. The better differentiated cells had abundant eosinophilic cytoplasm with prominent Nissl substance and were generally positive for neurofilament and variably positive for synaptophysin. The generally smaller and less-differentiated cells were infrequently positive for proliferating cell nuclear antigen and were negative for any neuronal and glial markers. No apparent glial differentiation of the immature tumor cells was detected. Based on morphologic and immunohistochemical features, the diagnosis of cerebral ganglioneuroblastoma was made. This neoplasm is very rare in all species, especially in the central nervous system, and has never been reported previously in this site in a dog.

Serotonin activates electrolyte transport via 5-HT2A receptor in rat colonic crypt cells.

The aim of this study was to demonstrate that 5-HT activates electrolyte transport directly via 5-HT2A receptor in rat colonic crypt cells. Patch-clamp whole cell recording was performed in isolated crypts to measure the 5-HT-induced changes in electrogenic K+ and Cl- currents. Superfusing 5-HT (10 microM) in the bath solution increased both K+ and Cl- currents, which were antagonized by the presence of ketanserin (1 microM), a selective 5-HT2A antagonist, in the bath solution. Mesulergine (1 microM) a 5-HT2A and 5-HT2C antagonist, had no inhibitory effect. Strong chelation of the intracellular Ca2+ by 5 mM BAPTA inhibited 5-HT-induced currents. 5-HT also failed to activate K+ and C1- currents in the presence of GDPbetaS (0.5 mM) in the pipette solution. Intracellular administration of GTPgammaS (0.1 mM) mimicked the stimulatory effect of 5-HT, that was inhibited by 5 mM BAPTA. H-7 (0.05 mM), an inhibitor of protein kinase C, A, and G, did not affect the currents. These data indicate that a G protein-coupled pathway is involved in the activation of electrolyte secretion via 5-HT2A receptor.

A mini Cl- channel sensitive to external pH in the basolateral membrane of guinea-pig parietal cells.

1. Voltage-independent whole-cell Cl- currents were recorded from both single, isolated parietal cells and parietal cells within gastric glands obtained from the fundus of guinea-pig stomach. 2. The Cl- currents were rapidly suppressed by a Cl- channel blocker, NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate), added to the (basolateral) bathing solution in a concentration-dependent manner with a half-maximal inhibition concentration of 12 microM. 3. The selectivity sequence among anions was I- > Br- > Cl- > F-, corresponding to Eisenman's sequence I. 4. The Cl- currents were independent of cytosolic Ca2+, cyclic AMP, cyclic GMP, GTP-gamma-S and cell volume, and were not affected by application of acid secretagogues, omeprazol, arachidonic acid or prostaglandin E2. 5. Reduction of pH in the (basolateral) bathing solution immediately inhibited the Cl- current with a pK (-log of KD) of 6.3, whereas changes in intracellular pH had no effect. 6. The single-channel conductance was estimated to be 0.46-0.6 pS by variance noise analysis during inhibition of whole-cell Cl- currents by NPPB or acidic pH. 7. It is concluded that pH-sensitive 'mini' Cl- channels, with a sub-picosiemens unitary conductance, exist in the basolateral membrane of guinea-pig parietal cells.

A maxi Cl- channel coupled to endothelin B receptors in the basolateral membrane of guinea-pig parietal cells.

1. To study endothelin (ET) receptors in guinea-pig stomach, ET-binding assays and in vitro autoradiography were performed on fundic cell suspensions and on sections of the fundus, respectively. ETA and ETB receptor subtypes were found to coexist in the parietal cells. 2. Endothelin 1 (ET-1) added to the (basolateral) bathing solution was found to activate noisy whole-cell Cl- currents within about 1 min in both single, isolated parietal cells and those within gastric glands obtained from the fundus. 3. ET-1-induced Cl- currents were rapidly blocked by a Cl- channel blocker (NPPB) added to the (basolateral) bathing solution in a concentration-dependent manner with a half-maximum inhibition concentration of 33 microM. 4. The anion selectivity sequence of the ET-1-induced conductance was I- > Br- > Cl- > F-, corresponding to Eisenman's sequence I. 5. Changes in extracellular pH between 5 and 8 did not affect the ET-1-induced activation of Cl- currents. 6. Similar activating effects were also observed with ET-3 and a specific ETB receptor agonist (IRL1620). An ETB receptor antagonist (IRL1720) prevented the ET-1 effect, whereas an ETA-selective antagonist (FR139317 or BQ123) failed to antagonize the ET-1 effect. 7. In the whole-cell mode, unitary Cl- channel events could be observed in association with ET-1-activated macroscopic currents. The single-channel conductances were around 200 and 350 pS at negative and positive membrane potentials, respectively. 8. It is concluded that gastric parietal cells of guinea-pig possess pH-insensitive 'maxi' Cl- channels coupled to ETB receptors in the basolateral membrane.

Evidence for two types of potassium current in rat choroid plexus epithelial cells.

The whole-cell patch-clamp technique was applied to rat choroid plexus epithelial cells. The resting membrane potential was -53 mV. The whole-cell conductance was mainly K+ selective, and the K+ current observed appeared to contain two distinct components. Depolarizing voltage pulses (more positive than 0 mV) evoked time-dependent outward currents which resembled delayed-rectifying K+ currents in other tissues. The current exhibited time-dependent activation and, at potentials more positive than 40 mV, slower time-dependent inactivation. The reversal potential measured by tail current analysis showed a shift of 43 mV for a tenfold increase in extracellular K+ concentration ([K+]o). The current was reduced by extracellular 5 mM Ba2+, 5 mM tetraethylammonium (TEA+), 5 mM Cs+ and 1 mM 4-aminopyridine (4-AP). In contrast, hyperpolarizing voltage pulses evoked time-independent, inward-rectifying currents. The reversal potential measured by voltage-ramp commands showed a shift of 42 mV for a tenfold increase in [K+]o. The chord conductance did not appear to increase with increasing [K+]o. The current was reduced by extracellular 5 mM Ba2+ and 0.5 mM Cs+, but not by 5 mM TEA+ or 1 mM 4-AP. These data suggest that two populations of K+ channel contribute to the conductance of choroid plexus epithelial cells.

Cl- current activation in choroid plexus epithelial cells involves a G protein and protein kinase A.

The involvement of GTP-binding proteins (G proteins) in the regulation of the Cl- conductance in rat choroid plexus epithelial cells was investigated, using the whole cell patch-clamp technique. Intracellular application of a nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 0.1-0.2 mM), evoked a transient increase in the Cl- conductance. The activated Cl- current exhibited inward rectification and was independent of time at hyperpolarizing or depolarizing voltage pulses. The effect of GTP gamma S was inhibited by a nonhydrolyzable GDP analogue, guanosine 5'-O-(2-thiodiphosphate) (2 mM), and by an inhibitor of protein kinase A, H-89, but was not affected by chelation of cytosolic Ca2+ with 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. GTP gamma S failed to activate the current when ATP was omitted from the pipette solution. Intracellular application of adenosine 3',5'-cyclic monophosphate (cAMP; 0.25 mM) or the catalytic subunit of protein kinase A activated a similar Cl- current. These results suggest that G proteins activate Cl- channels via a cAMP-dependent pathway in rat choroid plexus.

Activation of chloride currents in choroid plexus by GTP-gamma-S and cyclic-AMP.

The regulation of a Cl- conductance in the choroid plexus by G proteins was investigated using whole-cell patch clamp methods. A transient Cl- current was evoked when GTP-gamma-S (0.1 mM) was included in the electrode solution. The GTP-gamma-S-evoked current was only observed when 2 mM ATP was present in the electrode solution, and was inhibited in the presence of 2 mM GDP-beta-S. The activated current was shown to be Cl(-)-selective in ion replacement studies, and exhibited inward-rectification. The current was not inhibited when intracellular Ca2+ was strongly buffered with either EGTA or BAPTA, suggesting that the current was not activated by a change in intracellular Ca2+. The effects of GTP-gamma-S were mimicked, however, when cyclic AMP or the catalytic-subunit of protein kinase A were included in the electrode solution. These data suggest that G proteins activate Cl- channels via a cAMP-dependent pathway in rat choroid plexus.

Endothelin-induced activation of Cl- channels of guinea pig parietal cells.

The addition of endothelin-1 to the bath solution dose-dependently increased the whole-cell Cl- currents in single parietal cells isolated from guinea pig stomach. NPPB dose-dependently inhibited the current. Endothelin-3 was also effective, though less than endothelin-1.

Calcium-dependent chloride current activated by hyposmotic stress in rat lacrimal acinar cells.

We have identified a whole-cell Cl- current activated by hyposmotic stress in rat lacrimal acinar cells using the patch-clamp technique. Superfusion of isolated single cells with hyposmotic solution (80% of control osmolarity) caused a gradual increase of the current, which was reversed on return to the control solution. The current-voltage relationship showed outward rectification, and the current showed time and voltage dependence: slowly activated by depolarizing voltages and rapidly inactivated by hyperpolarizing voltages. The increase in current was not observed when intracellular Ca2+ was chelated with EGTA. It was also inhibited by the absence of extracellular Ca2+, or the presence of gadolinium ions (20 microM Gd3+). We conclude that in rat lacrimal acinar cells hyposmotic stress activates Ca(2+)-dependent Cl- channels as a result of Ca2+ influx through a Gd(3+)-sensitive pathway. The Cl- channels involved appear to be indistinguishable from those activated by muscarinic stimulation. The inhibitory effect of Gd3+ suggests that stretch-activated nonselective cation channels may be responsible for the Ca2+ influx.

Membrane capacitance increases induced by histamine and cyclic AMP in single gastric acid-secreting cells of the guinea pig.

During acid secretion, gastric parietal cells undergo profound morphological changes including formation of the apical secretory membrane. To examine the mechanism of histamine-induced increases in the apical membrane area at the single cell level, we monitored the membrane capacitance by applying a time-resolved phase-sensitive detection method to singly isolated parietal cells of guinea pig. A real-time increase in the membrane capacitance was detected within several min after stimulation with histamine. An H2-blocker (cimetidine), but not an H1-blocker (pyrilamine), inhibited the histamine response. Dibutyryl cyclic AMP mimicked the histamine effect. The capacitance response to histamine was sensitive to cytosolic Ca2+, temperature and N-ethylmaleimide. The histamine response was inhibited by intracellular application of a non-hydrolyzable ATP analog (AMP-PNP) and an isoquinolinesulfonamide derivative that works as an inhibitor of protein kinase A (H-8). These results indicate that in parietal cells, elevation of intracellular cyclic AMP induces exocytotic insertion of intracellular membranes into the plasma membrane, presumably by activating protein kinase A.

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig.

Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K+ (EK). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at near EK, indicating that both time-dependent and voltage-gated currents were due to K+ conductances. Both outward and inward K+ currents were suppressed by extracellular application of Ba2+, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs+ blocked the inward current but not the outward current. The conductance of inward K+ currents, but not outward K+ currents, became larger with increasing extracellular K+ concentration. A Ca(2+)-mobilizing acid secretagogue, carbachol, and a Ca2+ ionophore, ionomycin, brought about activation of another type of outward K+ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca2+ chelation. Quinine preferentially inhibited this K+ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K+ channels, inward rectifier and outward rectifier, and that the cells have Ca(2+)-activated K+ channels which might be involved in acid secretion under stimulation by Ca(2+)-mobilizing secretagogues.

[Prevention of alcohol-induced gastric mucosal injury by prostaglandin E-1].

Effects of ethanol and prostaglandin on the glycoprotein synthesis in the gastric mucus cells have been evaluated. The rat gastric mucosal cell were subjected to a short-term tissue culture in the presence of ethanol and prostaglandin E2 (Ornoprostil), using [3H]-proline, [3H]-palmitic acid, [3H]-galactosamine. Low concentration of ethanol (0.05-0.01 M) stimulated the glycoprotein synthesis, but higher concentration of ethanol (0.5-1.5 M) caused a marked reduction in the glycoprotein synthesis. Addition of prostaglandin E2, especially addition of prostaglandin prior to ethanol treatment, has improved the glycoprotein synthesis and secretion in the presence of ethanol. It was suggested that addition of prostaglandin resulted in the stabilization of the synthesis and secretion of the mucus glycoprotein in the presence of ethanol.