Distinct molecular mechanisms control levels of synaptic F‐actin

Polymerization of filamentous (F)‐actin at the neuronal synapse plays an important role in neuronal function. However, the regulatory mechanisms controlling the levels of synaptic actin remain incompletely understood. Here, I used established pharmacological blockers to acutely disrupt the function of actin polymerization machinery, then quantified their effect on synaptic F‐actin levels. Synaptic F‐actin was modestly decreased by inhibition of Arp2/3‐dependent actin branching. Blockade of formin‐dependent actin elongation resulted in an Arp2/3‐dependent increase in synaptic actin that could be mimicked by limited actin depolymerization. Limited actin depolymerization was also sufficient to reverse a decrease in synaptic F‐actin caused by prolonged blockade of synaptic NMDA‐type glutamate receptors. These results suggest that interplay between different actin polymerization pathways may regulate synaptic actin dynamics.     

Presynaptic effectors contributing to cAMP‐induced synaptic potentiation in Drosophila

cAMP analogs and activation of adenylyl cyclase by forskolin strongly potentiate synaptic transmission at the Drosophila neuromuscular junction. These effects are generally attributed to activation of cAMP‐dependent protein kinase. Recent reports on crustacean and mammalian synapses have implicated other cAMP‐dependent effectors in synaptic potentiation. Drosophila neuromuscular junctions were tested for effects of two known cAMP‐dependent effectors: hyperpolarization‐activated, cyclic nucleotide‐regulated channels (HCNCs) and guanine nucleotide exchange protein activated by cAMP (Epac). Forskolin‐induced enhancement of synaptic transmission was drastically reduced by a blocker of HCNCs, but not completely eliminated. A specific agonist for Epac modestly enhanced synaptic potentials. This agonist also stabilized their amplitudes in the presence of a blocker of HCNCs. The observations implicate HCNCs and Epac in cAMP‐dependent potentiation that does not require cAMP‐dependent protein kinase, indicating that additional previously unexplored factors contribute to synaptic plasticity in Drosophila. Genetic and molecular techniques available for Drosophila can be used to define the underlying molecular basis for cAMP‐dependent synaptic potentiation. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Presynaptic effectors contributing to cAMP-induced synaptic potentiation in Drosophila

cAMP analogs and activation of adenylyl cyclase by forskolin strongly potentiate synaptic transmission at the Drosophila neuromuscular junction. These effects are generally attributed to activation of cAMP-dependent protein kinase. Recent reports on crustacean and mammalian synapses have implicated other cAMP-dependent effectors in synaptic potentiation. Drosophila neuromuscular junctions were tested for effects of two known cAMP-dependent effectors: hyperpolarization-activated, cyclic nucleotide-regulated channels (HCNCs) and guanine nucleotide exchange protein activated by cAMP (Epac). Forskolin-induced enhancement of synaptic transmission was drastically reduced by a blocker of HCNCs, but not completely eliminated. A specific agonist for Epac modestly enhanced synaptic potentials. This agonist also stabilized their amplitudes in the presence of a blocker of HCNCs. The observations implicate HCNCs and Epac in cAMP-dependent potentiation that does not require cAMP-dependent protein kinase, indicating that additional previously unexplored factors contribute to synaptic plasticity in Drosophila. Genetic and molecular techniques available for Drosophila can be used to define the underlying molecular basis for cAMP-dependent synaptic potentiation.     

Reduced transition between open and inactivated channel states underlies 5HT increased I(Na+) in rat nociceptors

We previously demonstrated that activation of a 5HT(4) receptor coupled cAMP-dependent signaling pathway increases tetrodotoxin-resistant Na(+) current (I(Na)) in a nociceptor-like subpopulation of rat dorsal root ganglion cells (type 2). In the present study we used electrophysiology experiments and computer modeling studies to explore the mechanism(s) underlying the increase of I(Na) by 5HT. In electrophysiological experiments with type 2 dorsal root ganglion cells, 5HT increased peak I(Na) and the activation and inactivation rate, without significantly affecting the voltage dependency of activation or availability. Studies on the voltage dependency of channel availability, time course of removal of inactivation, and inactivation of evoked Na(+) currents suggested that there are at least two inactivation states of the Na(+) channel, one (I(fast)) that is induced and retrieved faster than the other (I(slow)). Long (1 s), but not short (60 or 100 ms), inactivating conditioning pulses (CPs) suppressed the 5HT-induced increase in I(Na). Computer modeling studies suggest that 5HT increased I(Na) mainly by decreasing the transition rate (k(OI1)) from an open state to I(fast). Furthermore, 5HT increased I(Na) activation and inactivation rates mainly by increasing the transition rate from closed to open (k(C3O)) and from I(fast) to I(slow) (k(I1I2)), respectively. The antagonism of the 5HT-induced increase in I(Na) by 1-s inactivation CPs may be due an enhancement of transitions from I(fast) to I(slow), via the increase in k(I1I2). This may deplete the pool of channels residing in I(fast), reducing the frequency of reopenings from I(fast), which offsets the increase in I(Na) produced by the reduction in k(OI1). The above findings fit well with previous studies showing that activation of the cAMP/PKA cascade simultaneously increases voltage sensitive tetrodotoxin-resistant Na(+) conductance and inactivation rate in nociceptors. The antagonism of the effects of 5HT by long inactivation CPs suggests that drugs designed to induce and/or stabilize the I(slow) state might be useful for reducing hyperalgesia produced by inflammatory mediators.     

L-plastin peptide activation of alpha(v)beta(3)-mediated adhesion requires integrin conformational change and actin filament disassembly

L-plastin (LPL) is a leukocyte actin binding protein previously implicated in the activation of the integrin alpha(M)beta(2) on polymorphonuclear neutrophils. To determine the role for LPL in integrin activation, K562 cell adhesion to vitronectin via alpha(v)beta(3), a well-studied model for activable integrins, was examined. Cell permeant versions of peptides based on the N-terminal sequence of LPL and the LPL headpiece domain both activated alpha(v)beta(3)-mediated adhesion. In contrast to adhesion induced by treatment with phorbol 12-myristate 13-acetate (PMA), LPL peptide-activated adhesion was independent of integrin beta(3) cytoplasmic domain tyrosines and was not inhibited by cytochalasin D. Also in contrast to PMA, LPL peptides synergized with RGD ligand or Mn(2+) for generation of a conformational change in alpha(v)beta(3) associated with the high affinity state of the integrin, as determined by binding of a ligand-induced binding site antibody. Although LPL and ligand showed synergy for ligand-induced binding site expression when actin depolymerization was inhibited by jasplakinolide, LPL peptide-induced adhesion was inhibited. Thus, both actin depolymerization and ligand-induced integrin conformational change are required for LPL peptide-induced adhesion. We hypothesize that the critical steps of increased integrin diffusion and affinity enhancement may be linked via modulation of the function of the actin binding protein L-plastin.     

Long-lasting synaptic potentials and the modulation of synaptic transmission.

Long-lasting postsynaptic potentials (PSPs) generated by decreases in membrane conductance (permeability) have been reported in many types of neurons. We investigated the possible role of such long-lasting decreases in membrane conductance in the modulation of synaptic transmission in the sympathetic ganglion of the bullfrog. The molecular basis by which such conductance-decrease PSPs are generated was also investigated. Synaptic activation of muscarinic cholinergic receptors on these sympathetic neurons results in the generation of a slow EPSP (excitatory postsynaptic potential), which is accompanied by a decrease in membrane conductance. We found that the conventional "fast" EPSPs were increased in amplitude and duration during the iontophoretic application of methacholine, which activates the muscarinic postsynaptic receptors. A similar result was obtained when a noncholinergic conductance-decrease PSP--the late-slow EPSP--was elicited by stimulation of a separate synaptic pathway. The enhancement of fast EPSP amplitude increased the probability of postsynaptic action potential generation, thus increasing the efficacy of impulse transmission across the synapse. Stimulation of one synaptic pathway is therefore capable of increasing the efficacy of synaptic transmission in a second synaptic pathway by a postsynaptic mechanism. Furthermore, this enhancement of synaptic efficacy is long-lasting by virtue of the long duration of the slow PSP. Biochemical and electrophysiological techniques were used to investigate whether cyclic nucleotides are intracellular second messengers mediating the membrane permeability changes underlying slow-PSP generation. Stimulation of the synaptic inputs, which lead to the generation of the slow-PSPs, increased the ganglionic content of both cyclic AMP and cyclic GMP. However, electrophysiological analysis of the actions of these cyclic nucleotides and the actions of agents that affect their metabolism does not provide support for such a second messenger role for either cyclic nucleotide.     

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.     

Arachidonic acid modulates Na+ currents by non-metabolic and metabolic pathways in rat cerebellar granule cells

AA (arachidonic acid), which possesses both neurotoxic and neurotrophic activities, has been implicated as a messenger in both physiological and pathophysiological processes. In the present study, we investigated the effects of both extracellular and intracellular application of AA on the activity of Na(V) (voltage-gated Na(+) channels) in rat cerebellar GCs (granule cells). The extracellular application of AA inhibited the resultant I(Na) (Na(V) current), wherein the current-voltage curve shifted to a negative voltage direction. Because this effect could be reproduced by treating the GCs with ETYA (eicosa-5,8,11,14-tetraynoic acid) or a membrane-impermeable analogue of AA, AA-CoA (arachidonoyl coenzyme A), we inferred that AA itself exerted the observed modulatory effects on I(Na). In contrast, intracellular AA significantly augmented the elicited I(Na) peak when the same protocol that was used for extracellular AA was followed. The observed I(Na) increase that was induced by intracellular AA was mimicked by the AA cyclo-oxygenase metabolite PGE(2) (prostaglandin E(2)), but not by ETYA. Furthermore, cyclo-oxygenase inhibitors decreased I(Na) and quenched AA-induced channel activation, indicating that the effect of intracellular AA on Na(V) was possibly mediated through AA metabolites. In addition, the PGE2-induced activation of I(Na) was mimicked by cAMP and quenched by a PKA (protein kinase A) inhibitor, a G(s) inhibitor and EP (E-series of prostaglandin) receptor antagonists. The results of the present study suggest that extracellular AA modulates Na(V) channel activity in rat cerebellar GCs without metabolic conversion, whereas intracellular AA augments the I(Na) by PGE(2)-mediated activation of cAMP/PKA pathways. These observations may explain the dual character of AA in neuronal pathogenesis.     

cAMP and sodium transport in the freshwater crayfish, Cherax destructor.

Crayfish in which sodium absorption was maximally stimulated had elevated levels of both cAMP and Na(+)-K(+)-ATPase activity in gill tissue. The concentration of cAMP and activity of Na(+)-K(+)-ATPase in gill tissue were monitored following transfer of crayfish from water containing 125 mmol x l(-1) Na to Na-free media. Both parameters were significantly elevated within 10 min of transfer to Na-free media and [cAMP] peaked between 1 and 2 h before falling transiently to the control level at 3 h. A second peak of [cAMP] and a further rise in Na(+)-K(+)-ATPase activity were evident 6 h after transfer and elevated levels were then maintained. The pattern observed was consistent with the existence of two separate mechanisms for the control of sodium absorption both of which stimulated the activity of Na(+)-K(+)-ATPase via elevation of the intracellular concentration of cAMP. The initial response was very rapid (<10 min) but of brief duration (1-2 h) and this mechanism appeared to be sensitive to changes in external ion levels. The second mechanism exhibited a much longer response time (3-6 h) and duration and was likely to be sensitive to changes in internal ion concentrations.     

The loading of neurotransmitters into synaptic vesicles

Classical (non-peptide) transmitters are stored into secretory vesicles by a secondary active transporter driven by a V-type H(+)-ATPase. Five vesicular neurotransmitter uptake activities have been characterized in vitro and, for three of them, the transporters involved have been identified at the molecular level using cDNA cloning and/or Caenorhabditis elegans genetics. These transporters belong to two protein families, which are both unrelated to the Na(+)-coupled neurotransmitter transporters operating at the plasma membrane. The two isoforms of the mammalian vesicular monoamine transporter, VMAT1 and VMAT2, are related to the vesicular acetylcholine transporter (VACHT), while a novel, unrelated vesicular inhibitory amino acid transporter (VIAAT), also designated vesicular GABA transporter (VGAT), is responsible for the storage of GABA, glycine or, at some synapses, both amino acids into synaptic vesicles. The observed effects of experimentally altered levels of VACHT or VMAT2 on synaptic transmission and behavior, as well as the recent awareness that GABAergic or glutamatergic receptors are not always saturated at central synapses, suggest a potential role of vesicular loading in synaptic plasticity.     

Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents

Oscillatory network activity at gamma frequencies is assumed to be of major importance in cortical information processing. Whereas the synaptic mechanisms of gamma oscillations have been studied in detail, the ionic currents involved at the cellular level remain to be elucidated. Here we show that in vitro gamma oscillations induced by muscarine require activation of M1 receptors on hippocampal CA3 pyramidal neurons and are absent in M1 receptor-deficient mice. M1 receptor activation depolarizes pyramidal neurons by increasing the mixed Na(+)/K(+) current I(h) and the Ca(2+)-dependent nonspecific cation current I(cat), but not by modulation of I(M). Our data provide important insight into the molecular basis of gamma oscillations by unequivocally establishing a novel role for muscarinic modulation of I(h) and I(cat) in rhythmic network activity.     

Biophysical properties and slow voltage-dependent inactivation of a sustained sodium current in entorhinal cortex layer-II principal neurons: a whole-cell and single-channel study.

The functional and biophysical properties of a sustained, or "persistent," Na(+) current (I(NaP)) responsible for the generation of subthreshold oscillatory activity in entorhinal cortex layer-II principal neurons (the "stellate cells") were investigated with whole-cell, patch-clamp experiments. Both acutely dissociated cells and slices derived from adult rat entorhinal cortex were used. I(NaP), activated by either slow voltage ramps or long-lasting depolarizing pulses, was prominent in both isolated and, especially, in situ neurons. The analysis of the gating properties of the transient Na(+) current (I(NaT)) in the same neurons revealed that the resulting time-independent "window" current (I(NaTW)) had both amplitude and voltage dependence not compatible with those of the observed I(NaP), thus implying the existence of an alternative mechanism of persistent Na(+)-current generation. The tetrodotoxin-sensitive Na(+) currents evoked by slow voltage ramps decreased in amplitude with decreasing ramp slopes, thus suggesting that a time-dependent inactivation was taking place during ramp depolarizations. When ramps were preceded by increasingly positive, long-lasting voltage prepulses, I(NaP) was progressively, and eventually completely, inactivated. The V(1/2) of I(NaP) steady state inactivation was approximately -49 mV. The time dependence of the development of the inactivation was also studied by varying the duration of the inactivating prepulse: time constants ranging from approximately 6.8 to approximately 2.6 s, depending on the voltage level, were revealed. Moreover, the activation and inactivation properties of I(NaP) were such as to generate, within a relatively broad membrane-voltage range, a really persistent window current (I(NaPW)). Significantly, I(NaPW) was maximal at about the same voltage level at which subthreshold oscillations are expressed by the stellate cells. Indeed, at -50 mV, the I(NaPW) was shown to contribute to >80% of the persistent Na(+) current that sustains the subthreshold oscillations, whereas only the remaining part can be attributed to a classical Hodgkin-Huxley I(NaTW). Finally, the single-channel bases of I(NaP) slow inactivation and I(NaPW) generation were investigated in cell-attached experiments. Both phenomena were found to be underlain by repetitive, relatively prolonged late channel openings that appeared to undergo inactivation in a nearly irreversible manner at high depolarization levels (-10 mV), but not at more negative potentials (-40 mV).     

IP3-mediated octopamine-induced synaptic enhancement of crayfish LG neurons

The biogenic amines, octopamine and serotonin, modulate the synaptic activity of the lateral giant interneuron (LG) circuitry of the crayfish escape behavior. Bath application of both octopamine and serotonin enhances the synaptic responses of LG to sensory stimulation. We have shown previously (Araki et al. J Neurophysiol 94:2644-2652, 2005) that a serotonin-induced enhancement of the LG response was mediated by an increase in cAMP levels following activation of adenylate cyclase; however, octopamine acts independently. Here, we clarify how octopamine enhances the LG response during sensory stimulation using physiological and pharmacological analyses. When phospholipase C inhibitor U-73122 was directly injected into the LG before biogenic amine application, it abolished the enhancing effect of octopamine on direct sensory input to the LG, but did not block indirect input via sensory interneurons or the effect of serotonin. Direct injection of IP(3), and its analogue adenophostin A, into the LG increased the synaptic response of the LG to sensory stimulation. Thus, IP(3) mediates octopamine-induced synaptic enhancement of the LG, but serotonin acts independently. These results indicate that both octopamine and serotonin enhance the synaptic responses of the LG to sensory stimulation, but that they activate two different signaling cascades in the LG.     

Evidence that 3-hydroxyglutaric acid interacts with NMDA receptors in synaptic plasma membranes from cerebral cortex of young rats

Neurological symptoms are common in patients with glutaric acidemia type I (GA-I). Although the pathophysiology of this disorder is not yet fully established, 3-hydroxyglutaric acid (3-HGA), which accumulates in affected patients, has recently been demonstrated to be excitotoxic to embryonic chick and neonatal rat neurons probably via NMDA glutamate receptors. In the present study, we investigated the in vitro effects of 3-HGA on the [(3)H]glutamate and [(3)H]MK-801 (dizocilpine) binding to rat synaptic plasma membranes from cerebral cortex of young rats in order to elucidate the interactions of 3-HGA with glutamate receptors and its possible contribution to the in vitro excitotoxic properties of 3-HGA. 3-HGA (10-100 microM) significantly decreased Na(+)-dependent (up to 62%) and Na(+)-independent (up to 30%) [(3)H]glutamate binding to synaptic membranes, reflecting a possible competition between glutamate and 3-HGA for the glutamate transporter and receptor sites, respectively. Since a decrease in Na(+)-independent glutamate binding might represent an interaction of 3-HGA with glutamate receptors, we next investigated whether 3-HGA interacts with NMDA receptors by adding NMDA alone or combined with 3-HGA and measuring Na(+)-independent [(3)H]glutamate binding to synaptic membranes (binding to receptors). We verified that 3-HGA and NMDA, at 10 and 100 microM concentrations, decreased glutamate binding by up to 20 and 45%, respectively, and that the simultaneous addition of both substances did not provoke an additive effect, implying that they bind to NMDA receptors at the same site. Furthermore, the binding of the NMDA-channel blocker [(3)H ]MK-801 was significantly increased (approximately 32-40%) by 10 and 100 microM 3-HGA, implying that 3-HGA was able to open the NMDA channel allowing MK-801 binding, which is a characteristic of NMDA agonists. On the other hand, glutamate had a much higher stimulatory effect on this binding (180% increase), reflecting its strong NMDA agonist property. Furthermore, the simultaneous addition of 3-HGA and glutamate provoked an additive stimulatory effect on [(3)H]MK-801 binding to the NMDA receptor. These data indicate that, relatively to glutamate, 3-HGA is a weak agonist of NMDA receptors. Finally, we demonstrated that 3-HGA provoked a significant increase of extracellular calcium uptake by cerebral cortex slices, strengthening therefore, the view that 3-HGA activates NMDA receptors. The present study therefore, demonstrates at the molecular level that 3-HGA modulates glutamatergic neurotransmission and may explain previous findings relating the neurotoxic actions of this organic acid with excitotoxicity.     

Glutaraldehyde fixation of the cAMP-dependent Na+/H+ exchanger in trout red cells

It has been shown that the addition of a beta-adrenergic catecholamine to a trout red blood cell suspension induces a 60-100-fold increase of sodium permeability resulting from the activation of a cAMP-dependent Na+/H+ antiport. Subsequent addition of propranolol almost instantaneously reduces the intracellular cAMP concentration, and thus the Na permeability, to their basal values (Mahé et al., 1985). If glutaraldehyde (0.06-0.1%) is added when the Na+/H+ exchanger is activated after hormonal stimulation, addition of propranolol no longer inhibits Na permeability: once activated and fixed by glutaraldehyde, the cAMP dependence disappears. Glutaraldehyde alone causes a rapid decrease in the cellular cAMP concentration. In its fixed state the antiporter is fully amiloride sensitive. The switching on of the Na+/H+ exchange by cAMP is rapidly (2 min) followed by acute but progressive desensitization of the exchanger (Garcia-Romeu et al., 1988). The desensitization depends on the concentration of external sodium, being maximal at a normal Na concentration (145 mM) and nonexistent at a low Na concentration (20 mM). If glutaraldehyde is added after activation in nondesensitizing conditions (20 mM Na), transfer to a Na-rich medium induces only a very slight desensitization: thus the fixative can "freeze" the exchanger in the nondesensitizing conformation. NO3- inhibits the activity of the cAMP-dependent Na+/H+ antiporter of the trout red blood cell (Borgese et al., 1986). If glutaraldehyde is added when the cells are activated by cAMP in a chloride-containing medium, the activity of the exchanger is no longer inhibited when Cl- is replaced by NO3-. Conversely, after fixation in NO3- medium replacement of NO3- by Cl- has very little stimulatory effect. This indicates that the anion dependence is not a specific requirement for the exchange process but that the anion environment is critical for the switching on of the Na+/H+ exchanger and for the maintenance of its activated configuration.     

Activation of Rho GTPases by synaptic transmission in the hippocampus

Ras-related GTPases of the Rho family, such as RhoA and RhoB, are well-characterised mediators of morphological change in peripheral tissues via their effects on the actin cytoskeleton. We tested the hypothesis that Rho family GTPases are involved in synaptic transmission in the CA1 region of the hippocampus. We show that GTPases are activated by synaptic transmission. RhoA and RhoB were activated by low frequency stimulation, while the induction of long-term potentiation (LTP) by high frequency stimulation was associated with specific activation of RhoB via NMDA receptor stimulation. This illustrates that these GTPases are potential mediators of synaptic transmission in the hippocampus, and raises the possibility that RhoB may play a role in plasticity at hippocampal synapses during LTP.     

Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS

In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PGE(1) in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, cholera toxin, and PGE(1) induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. Pretreatment with the Src kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2) or geldanamycin or the PKA inhibitor Rp-cAMPS only partly inhibited isoproterenol- or PGE(1)-induced actin depolymerization. In contrast, PP2 and geldanamycin did not inhibit forskolin-induced actin depolymerization, and AG-213 (an EGF receptor tyrosine kinase inhibitor) did not inhibit isoproterenol- or PGE(1)-induced actin depolymerization. PI3 kinase or MAP kinase inhibition did not inhibit isoproterenol-induced actin depolymerization. Moreover, isoproterenol but not forskolin induced tyrosine phosphorylation of an Src family member at position 416. These results further confirm that both PKA-dependent and PKA-independent pathways mediate actin depolymerization in human airway smooth muscle cells and that the PKA-independent pathway by which isoproterenol induces actin depolymerization in human airway smooth muscle cells involves Src protein tyrosine kinases and the G(s) protein.