Tyrosine Kinase Inhibitors Tyrphostin A23 Research Articles

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Angiotensin II type 1 receptor mediates partially hyposmotic-induced increase of I Ks current in guinea pig atrium

A repolarizing conduction in the heart augmented by hyposmotic or mechanically induced membrane stretch is the slow component of delayed rectifier K(+) current (I (Ks)). I (Ks) upregulation is recognized as a factor promoting appearance of atrial fibrillation (AF) since gain-of-function mutations of the channel genes have been detected in congenital AF. Mechanical stretch activates angiotensin II type 1 (AT(1)) receptor in the absence of its physiological ligand angiotensin II. We investigated the functional role of AT(1) receptor in I (Ks) enhancement in hyposmotically challenged guinea pig atrial myocytes using the whole-cell patch-clamp method. In atrial myocytes exposed to hyposmotic solution with osmolality decreased to 70% of the physiological level, I (Ks) was enhanced by 84.1%, the duration of action potential at 90% repolarization (APD(90)) was decreased by 16.8%, and resting membrane potential was depolarized (+4.9 mV). The hyposmotic-induced effects on I (Ks) and APD(90) were significantly attenuated by specific AT(1) receptor antagonist candesartan (1 and 5 muM). Pretreatment of atrial myocytes with protein tyrosine kinase inhibitors tyrphostin A23 and A25 suppressed but the presence of tyrosine phosphatase inhibitor orthovanadate augmented hyposmotic stimulation of I (Ks). The above results implicate AT(1) receptor and tyrosine kinases in the hyposmotic modulation of atrial I (Ks) and suggest acute antiarrhythmic properties of AT(1) antagonists in the settings of stretch-related atrial tachyarrhythmias.

Signaling pathways in ascidian oocyte maturation: Effects of various inhibitors and activators on germinal vesicle breakdown

The Ascidiacea, the invertebrate chordates, includes three orders; the Stolidobranchia is the most complex. Until the present study, the onset of oocyte maturation (germinal vesicle breakdown) had been investigated in only a single pyurid (Halocynthia roretzi), in which germinal vesicle breakdown (GVBD) begins when the oocyte contacts seawater (SW); nothing was known about internal events. This study strongly suggests the importance of protein phosphorylation in this process. Herdmania pallida (Pyuridae) functions like H. roretzi; GVBD occurs in SW. Oocytes of Cnemidocarpa irene (Styelidae) do not spontaneously undergo GVBD in SW but must be activated. Herdmania oocytes are inhibited from GVBD by pH 4 SW and subsequently activated by mastoparan (G-protein activator), A23187 (Ca2+ ionophore) or dimethylbenzanthracene (tyrosine kinase activator). This requires maturation promoting factor (MPF) activity; cyclin-dependent kinase inhibitors roscovitine and olomoucine are inhibitory. It also entails dephosphorylation as demonstrated by the ability of the phosphatase inhibitor vitamin K3 to inhibit GVBD. GVBD is also inhibited by the tyrosine kinase inhibitors tyrphostin A23 and genistein, and LY-294002, a phosphatidylinositol-3-kinase inhibitor previously shown to inhibit starfish GVBD. LY-294002 inhibits strongly when activation is by mastoparan or ionophore but not when activated by dimethylbenzanthracene (DMBA). The DMBA is hypothesized to phosphorylate a phosphatase directly or indirectly causing secondary activation, bypassing inhibition.

Regulation of cardiac Na–Ca exchange activity by selective tyrosine kinase inhibition

British Journal of Pharmacology (2004) 143, 929–930. doi:10.1038/sj.bjp.0706009

Activation of Background Membrane Conductance by the Tyrosine Kinase Inhibitor Tyrphostin A23 and Its Inactive Analog Tyrphostin A1 in Guinea Pig Ventricular Myocytes

The effects of the tyrosine kinase (TK) inhibitor tyrphostin A23 and its inactive analog tyrphostin A1 on background membrane conductance were investigated in guinea pig ventricular myocytes. TK-inhibiting A23 reversibly increased membrane conductance under conditions designed to minimize Na+, Ca2+, K+, and Na+-K+ pump currents. Similar stimulatory action was obtained with TK-inactive A1. The tyrphostin-induced current was inhibited by omitting external Na+ or Ca2+, suppressed by chelating internal Ca2+, blocked by external Cd2+ and Ni2+, and insensitive to changes in internal Cl- concentration. We conclude that tyrphostins have a direct, TK-independent action that increases membrane conductance probably by stimulating Na+-Ca2+ exchange.

ET(A) receptors are the primary mediators of myofilament calcium sensitization induced by ET-1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway.

We have investigated the possibility that ET-1 can induce an increase in myofilament calcium sensitivity in pulmonary artery smooth muscle. Arterial rings were permeabilized using alpha-toxin (120 microg ml(-1)), in the presence of A23187 (10 microM) to 'knock out' Ca2+ stores, and pre-constricted with pCa 6.8 (buffered with 10 mM EGTA). In the presence of this fixed Ca2+ concentration, 1 microM ET-1 induced a sustained, reversible constriction of 0.15 mN. Pulmonary arterial rings were freeze-clamped at the peak of the induced constriction (time matched). Subsequent densitometric analysis revealed that ET-1 (1 microM) increased the level of phosphorylated myosin light chains by 34% compared to an 11% increase in the presence of pCa 6.8 alone. In contrast to ET-1, the selective ET(B) receptor agonist Sarafotoxin S6C (100 nM) failed to induce a significant constriction. The constriction induced by 1 microM ET-1 was reversibly inhibited when the preparation was preincubated (15 min) with the ETA receptor antagonist BQ 123 (100 microM). The constriction measured 0.13 mN in the absence and 0.07 mN in the presence of 100 microM BQ 123. In contrast, the constriction induced by 1 microM ET-1 measured 0.19 mN in the absence and 0.175 mN following a 15 min pre-incubation with the ET(B) antagonist BQ 788 (100 microM). The constriction induced by 1 microM ET-1 measured 0.14 mN in the presence and 0.13 mN following pre-incubation with the tyrosine kinase inhibitor Tyrphostin A23 (100 microM). We conclude that ET-1 induced an increase in myofilament calcium sensitivity in rat pulmonary arteries via the activation of ET(A) receptors and by a mechanism(s) independent of tyrosine kinase.

Endothelin-1 Stimulates Hydrolysis of Phosphatidylcholine by Phospholipases C and D in Intact Rat Mesenteric Arteries

A characteristic of endothelin-1 (ET-1)-induced contraction is the prolonged duration of the response. Phosphatidylcholine (PC) hydrolysis has been implicated in sustained agonist-induced effects through the formation of the second messengers phosphatidic acid (PA) and diacylglycerol (DAG). Therefore, we have investigated the activation of PC-phospholipase D (PLD) and PC-phospholipase C (PLC) in intact rat mesenteric small arteries stimulated with ET-1 and determined whether PC-derived DAG is necessary for ET-1-induced contraction. PLD activity, measured as phosphatidylethanol (PEt) production in vessels labelled with [³H] or [¹⁴C]myristate, was increased in a concentration- and time-dependent manner following ET-1 stimulation, as were [³H]PA levels, peaking at 10 min (ET-1 100 nM) and remaining above control levels for up to 20 min. Inclusion of 0.5% ethanol during ET-1 stimulation reduced [³H]PA levels, but did not alter the time course of formation. In addition, [¹⁴C]choline release was increased, confirming PLD-mediated PC hydrolysis. In contrast, DAG levels, measured by [³H]myristate labelling and mass assay, increased transiently at 5 min of ET-1 stimulation only. Analysis of the subclasses of DAG demonstrated an increase in all types of DAG without any enrichment with arachidonate-containing species, indicating PC, not inositol lipid hydrolysis was the source of DAG. The PC-PLC inhibitor D609, 2.5 µg/ml, completely abolished the ET-1-induced increase in [¹⁴C]DAG without affecting the increase in [¹⁴C]PA, PLD activity or the contractile response. In [¹⁴C]choline-labelled vessels, [¹⁴C]phosphocholine levels were increased by ET-1 with a similar time course to DAG production. Removal of extracellular Ca²⁺ and addition of 0.1 or 2 mM EGTA completely inhibited ET-1-stimulated PLD activity. The tyrosine kinase inhibitor tyrphostin A23 (100 µM) abolished ET-1-induced [³H]PA, [³H]PEt and [¹⁴C]DAG increases, whilst the negative analogue A1 was without effect. These data suggest that ET-1 couples via a calcium-dependent tyrosine kinase mechanism to PLD and PC-PLC in vascular smooth muscle. PC-derived DAG did not appear to be necessary for the contractile response, whereas sustained formation of PA, generated by PLD activity, implies that this lipid second messenger could be involved in the prolonged contractile response to this peptide.

Synergistic cytotoxicity, apoptosis and protein-linked DNA breakage by etoposide and camptothecin in human U87 glioma cells: dependence on tyrosine phosphorylation.

In this study, simultaneous administration of certain inhibitors of topoisomerase I and topoisomerase II produced synergistic cytotoxicity in a series of human glioma cell lines. Camptothecin (CPT) and etoposide (VP-16) produced combination indices (CI) <1.0 in all glioma cell lines tested, including those that were relatively resistant to the two topoisomerase inhibitors individually. In contrast, CPT and VP-16 produced additive cytotoxicity in HT-29 and SW-620 colon carcinoma cell lines. To explore the molecular basis for synergy in glioma cells, we focused on one glioma cell line (U87) in which even sub-cytotoxic doses of CPT potentiated the action of VP-16. Except for genistein (a topo II agent with tyrosine kinase inhibitory function), all topo II inhibitors tested (doxorubicin, ellipticine, and m-AMSA) were synergistic with CPT. While CPT and VP-16 produced cytotoxicity and protein-linked DNA breaks (PLDB) that were supra-additive in U87 glioma cells, CPT and genistein produced additive results. Pretreatment of U87 cells with the tyrosine kinase inhibitor tyrphostin-A23 or the tyrosine phosphatase activator O-phospho-L-tyrosine (OPLT) reduced combination PLDB from synergistic to additive levels, but had no effect on the formation of PLDB induced by either CPT or VP-16 alone. CPT and VP-16 also produced a synergistic accumulation of sub-G0 (apoptotic) cells which was blocked by tyrphostin-A23. No significant increase in topoisomerase protein levels could be detected in response to combination treatment. Thus, synergistic effects between topoisomerase I and topoisomerase II inhibitors in U87 glioma cells may depend upon phosphorylation of cellular proteins other than the topoisomerases themselves.

Role of tyrosine kinase in erythrocyte lysate-induced contraction in rabbit cerebral arteries.

This study was undertaken to explore whether erythrocyte lysate, a proposed cause of vasospasm, produces vasoconstriction by activation of tyrosine kinase in rabbit cerebral arteries. Isometric tension was used to monitor contractions in rabbit basilar arteries induced by erythrocyte lysate, 5-hydroxytryptamine (5-HT), or KCl in the absence or presence of tyrosine kinase inhibitors. Erythrocyte lysate, 5-HT, or KCl produced concentration-dependent contractions in rabbit basilar arteries. Preincubation with the tyrosine kinase inhibitors tyrphostin A23 and genistein (30 and 100 microM), but not diadzein, an inactive analog of genistein, attenuated significantly the contraction induced by erythrocyte lysate (p < 0.05). Tyrphostin A23, genistein, and diadzein (30 microM) failed to reduce the contraction caused by 5-HT. Genistein, but not tyrphostin A23 or diadzein (30 microM), attenuated significantly the contraction induced by KCl (p < 0.05). In another series, arterial rings were initially contracted with erythrocyte lysate, 5-HT, or KCl and the relaxant effect of genistein was then tested. Genistein relaxed rabbit basilar arteries that had been contracted by exposure to erythrocyte lysate, 5-HT, or KCl (30-100 microM; p < 0.05). These data indicate that tyrosine kinase may play a role in the regulation of cerebral arterial contraction and tyrosine kinase inhibitors may be useful in the management of cerebral vasospasm.

Activin-induced inhibin alpha-subunit production by rat granulosa cells requires endogenous insulin-like growth factor-I.

Inhibin-alpha subunit (Inh-alpha) gene expression is important for granulosa cell (GC) differentiation and prevention of ovarian tumorigenesis. Studies on Inh-alpha regulation have implicated activin and insulin-like growth factor-I (IGF-I) in the mechanisms of expression. Here we present evidence that endogenously produced IGF-I plays an obligatory role in activin-induced Inh-alpha production. Primary cultures of rat GC were incubated with increasing concentrations of various regulatory molecules, and the levels of Inh-alpha protein and its mRNA were measured in conditioned medium and cells, respectively. Recombinant activin A stimulated Inh-alpha expression, and the effects were dose- and time-dependent. The receptor tyrosine kinase inhibitor tyrphostin A23 caused a dose-dependent inhibition of activin-dependent Inh-alpha expression, whereas the inactive isomer, A63, had no effect. The stimulatory effect of activin was also blocked in a dose-dependent manner by added IGF binding protein-4 or -5, and the effects were reversed by IGF-I. Moreover, increasing concentrations of an anti-IGF-I antibody had a similar inhibitory effect on activin-stimulated Inh-alpha expression. Collectively, these results suggest, for the first time, that endogenously produced IGF-I is required for activin stimulation of Inh-alpha expression in cultured rat GC.