Blocking Protein Kinase Research Articles

Interaction of the GTP-binding protein Gi2 with a protein kinase A-like kinase in mouse fibroblasts.

We have previously shown that the GTP-binding protein, Gi2 of mouse Balb/c3T3 cells is linked to a serine kinase which phosphorylates the alpha-subunit of Gi itself. In this report we show that Gi is coupled to a second protein kinase. This kinase does not phosphorylate G but phosphorylates another protein bound non-covalently to G. Phosphorylation of the Gi-linked protein induces its release from Gi. Kinase activity is slightly enhanced by GTPyS, suggesting that this kinase may be physiologically regulated by Gi. In an attempt to identify the kinase we have examined the effect of peptide substrates and inhibitors on kinase activity. We found that the protein kinase A inhibitory peptide, PK1 5-24, inhibited the kinase activity, but at concentrations above those usually required to block protein kinase A. The protein kinase A substrate peptide, kemptide, acted as a substrate of the kinase, and was an inhibitor of the phosphorylation of the Gi-linked protein. However, a protein kinase A, catalytic subunit antibody failed to react with any proteins linked to Gi., A protein kinase C inhibitory peptide had no effect on phosphorylation of the Gi-linked protein. Thus, the identity of this kinase has not been resolved, but it may form part of the signalling system of activated Gi in fibroblasts.

Role of CFTR in chloride secretion across human tracheal epithelium.

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.

Theoretical analysis of the regulation of interferon expression during priming and blocking

Interferon superinduction, in the case of cell pretreatment with low doses of interferon (priming), may be explained by activation of 2′,5′-oligoadenylate synthetase and endonuclease L, since the latter, as expected, leads to a more rapid amplification of the standard scheme of interferon induction based on the antirepression mechanism. In the given case, endonuclease L will further increase the degradation rates for messages, which encode repressor proteins controlling interferon gene expression. Under ordinary induction, these messages are destroyed only by short-lived nuclease activated by double-stranded RNA. Cell pretreatment with high doses of interferon (blocking) considerably increases the concentrations of protein kinase and 2′,5′-oligoadenylate synthetase in the cell. However, it seems that during blocking protein kinase plays the main role in inhibition of interferon synthesis, and this leads to almost complete depression of translation in the cell. When protein kinase is not sufficiently activated, blocking does not occur since treatment of cells with high concentrations of interferon does not hinder priming induced by 2′,5′-oligoadenylate synthetase and endonuclease L. The proposed model is consistent with the findings that both interferon-treated primed and blocked cells are able to produce interferon more rapidly than normal cells. The analysis, based on a computer simulation model, suggests that priming and blocking of interferon may be based on processes controlling its induction and antiviral activity.

Effects of proctolin on contractions, membrane resistance, and non- voltage-dependent sarcolemmal ion channels in crustacean muscle fibers

The neuropeptide proctolin in nanomolar concentrations enhances the contraction of crustacean muscle fibers manyfold. The cellular mechanisms underlying this potentiation were investigated in single, isolated, fast-contracting abdominal extensor muscle fibers of a small crustacean, the marine isopod Idotea baltica. Force measurements and current-clamp experiments revealed two actions of proctolin on the muscle fibers. In half of the preparations, proctolin (10(-9)-10(-6) M) increased the fiber's input resistance by up to 25%. In about one-fourth of the preparations, proctolin induced all-or-none action potentials in response to depolarizing current pulses in muscle fibers that showed graded electric responses under control conditions. In both cases, proctolin potentiated the peak force of muscle contractions (between 1.5- and 18-fold for 5 x 10(-9) M proctolin). Proctolin affected neither the membrane resting potential nor the threshold for excitation-contraction coupling. Using cell-attached patches on the sarcolemmal membrane, we identified non-voltage-dependent ion channels which contribute to the passive membrane properties of the muscle fibers. A 53 +/- 6 pS channel had its reversal potential near rest and carried outward current at depolarized potentials with physiological saline in the recording pipette. With isotonic K+ saline in the patch pipette, the reversal potential was +85 +/- 12 mV depolarized from the resting potential and single-channel conductances ranged from 36 to 166 pS. Proctolin modulated the activity of all these putative K+ channels by reducing the number of functionally active channels. The effects of proctolin on force of contraction, input resistance, and single-channel activity were mimicked by a membrane-permeating analog of cAMP. Conversely, a monothio analog of cAMP (Rp-cAMPS), a blocker of protein kinase A activity, substantially decreased the membrane input resistance of the muscle fibers. The results suggest that activation of the cAMP signal pathway and phosphorylation of non-voltage-dependent K+ channels by protein kinase A are involved in the potentiation of contractions by proctolin in the muscle fibers of this crustacean.

Limitation of myocardial infarct size by adenosine A1 receptor activation is abolished by protein kinase C inhibitors in the rabbit

The aim was to test whether infarct size limitation by adenosine A1 receptor activation is mediated by protein kinase C. In the first series of experiments, myocardial infarction was induced in rabbits under pentobarbitone anaesthesia by 30 min coronary artery occlusion and 3 h reperfusion. Rabbits were pretreated with no drugs (control). 1 mg.kg-1 of R(-)N6-2-phenylisopropyl adenosine (PIA), 50 micrograms.kg-1 of staurosporine (Stauro), 2.5 mg.kg-1 of polymyxin B (PolyB), and a combination of PIA and either Stauro or PolyB. Infarct size and the area at risk were determined by tetrazolium staining and fluorescent particles, respectively. In the second series of experiments, the inotropic response to 4 beta-phorbol 12-myristate 13-acetate (PMA) was assessed in rabbits with and without pretreatment using the same doses of Stauro and PolyB as those in the first series of experiments. Infarct size expressed as percent of area at risk (%IS/AR) was significantly smaller in the PIA treated group than in the control: %IS/AR = 19.0(SEM 2.4)% v 37.7(4.0)%, P < 0.05. However, %IS/AR in the groups given Stauro [35.0(4.2)%], PolyB [37.9(3.2)%], PIA plus Stauro [34.9(3.9)%], and PIA plus PolyB [36.3(3.3)%] did not differ from the control value. PMA at the dose of 0.02 and 0.05 micrograms.kg-1 caused a dose dependent increase of the left ventricular dP/dtmax in the untreated rabbits. Such a positive inotropic response to PMA was not detected in rabbits pretreated with Stauro or PolyB, suggesting that the doses of the protein kinase C inhibitors were appropriate to block protein kinase C in the heart. Infarct size limitation by A1 receptor stimulation is mediated by activation of protein kinase C in pentobarbitone anaesthetised rabbits.

The action of 5-HT on calcium-dependent potassium channels and on the spinal locomotor network in lamprey is mediated by 5-HT 1A-like receptors

5-HT has a powerful modulatory action on the firing properties of single neurons as well as on locomotor activity. In lamprey, 5-HT increases the neuronal firing frequency in spinal neurons by reducing the conductance in Ca 2+-dependent K + channels (K Ca) underlying the slow afterhyperpolarization (sAHP), and it also lowers burst frequency of the spinal locomotor network. To elucidate which type of 5-HT receptor mediates these effects, different specific receptor agonists and antagonists were applied during intracellular current lamp recordings and during NMDA-induced fictive locomotion in the lamprey spinal cord in vitro preparation. The 5-HT 1A receptor agonist 8-OH-DPAT ((±)-8-hydroxy-dipropylaminotetralin hydrobromide), the 5-HT 1 receptor agonist 5-CT (5-car☐yamidotryptamine maleate) and the 5-HT 2 receptor agonist α-CH 3-5-HT (α-methylserotonin maleate) all reproduced the actions of 5-HT at both the cellular and the network levels. The effects of all agonists were completely or partially blocked by the 5-HT 1A and 5-HT 2 receptor antagonist spiperone (spiroperidol hydrochloride) while selective 5-HT 2 receptor antagonists were ineffective. The selective 5-HT 1A receptor antagonist S(−)-UH301 (S(−)-5-fluoro-8-hydroxy-dipropylaminotetralin hydrochloride) also counteracted the effect of 5-HT on the sAHP. 5-HT 3 and 5-HT 4 receptor agonists and antagonists were without effects. The intracellular coupling mechanism was not sensitive to pertussis toxin nor to the cAMP dependent protein kinase blocker (Rp)-cAMPS. These results indicate that the intracellular coupling mechanism is not likely to be due to a down regulation of adenylate cyclase activity or through a direct modulation of K + channels, as is common for 5-HT 1 receptors. The present results taken together with previous data indicates that the receptor responsible for the effects of 5-HT on the sAHP, and on the locomotor pattern generator in lamprey shares certain features, but is not identical to the mammalian 5-HT 1A receptor.

CGMP antagonizes angiotensin-mediated phosphatidylcholine hydrolysis and C kinase activation in mesangial cells

Previous studies from this laboratory have shown that in cultured rat mesangial cells (MC), angiotensin II (ANG II) mediates its effects via activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PC-PLD). In addition, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating maneuvers that stimulate particulate and soluble guanylate cyclase [atrial natriuretic factor (ANF) and sodium nitroprusside (SNP), respectively] antagonize ANG II-mediated PI-PLC activation. The current study explored whether cGMP impairs ANG II-mediated PC-PLC and PLD activity. The ANG II-stimulated release of the water-soluble metabolites of PC breakdown (phosphorylcholine and choline) was blocked by ANF and SNP. ANG II-stimulated phosphatidic acid and phosphatidylethanol formation were significantly reduced by ANF and SNP, confirming that cGMP blunted PLD activity. The inhibitory effect of cGMP on PLD could be reversed by N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide, a blocker of cGMP-dependent protein kinase. In parallel experiments, ANF and SNP abrogated sustained diacylglycerol (DAG) accumulation derived from ANG II stimulation of PC hydrolysis, confirming that cGMP diminished PC-PLC activity. Inhibition of PC-derived DAG accumulation by cGMP was associated with a concomitant decrement in ANG II-mediated translocation of protein kinase C (PKC) activity from the cytosol to the membrane. In summary, in MC, cGMP antagonizes ANG II-mediated PC hydrolysis, DAG formation, and PKC activation. We propose that cGMP-mediated inhibition of phospholipid metabolism and PKC translocation plays an important role in MC vasorelaxation.

Heat-Stable Toxin from &lt;i&gt;Escherichia col&lt;/i&gt;&lt;i&gt;i&lt;/i&gt; Activates Chloride Current via cGMP-Dependent Protein Kinase

Heat-stable toxin (STa) increases cyclic GMP (cGMP) in isolated intestinal cells and in T84 cells, a colonic secretory cell line. Whole-cell current recordings from patch clamp experiments show identical properties for currents activated by either STa or the cystic fibrosis transmembrane conductance regulator (CFTR) channel. STa-activated currents display a linear current-voltage relationship and a relative permeability sequence of Br > Cl > I. STa or 8-Br-cGMP-activated currents remain when 20 µ<i>M</i> Walsh inhibitor, a blocker of protein kinase A (PKA), is added in the pipette, suggesting that cGMP-dependent protein kinase (PKG) activates the currents. Intracellular addition of Rp-8-Br-cGMP, an agent that activates PKGII and inhibits PKGI and PKA, causes induction of a chloride conductance identical to that stimulated by STa. We conclude that STa activates CFTR by phosphorylation with cGMP-dependent protein kinase.

Interferons block protein kinase C-dependent but not-independent activation of Raf-1 and mitogen-activated protein kinases and mitogenesis in NIH 3T3 cells.

Interferons (IFNs) exert antiproliferative effects on many types of cells. The underlying molecular mechanism, however, is unclear. One possibility is that IFNs block growth factor-induced mitogenic signaling, which involves activation of Ras/Raf-1/MEK/mitogen-activated protein kinase. We have tested this hypothesis by using HER14 cells (NIH 3T3 cell expressing both platelet-derived growth factor [PDGF] and epidermal growth factor [EGF] receptors) as a model system. Our studies showed that IFNs (alpha/beta and gamma) blocked PDGF-and phorbol ester- but not EGF-stimulated DNA synthesis and cell proliferation. While the ligand-stimulated receptor tyrosine phosphorylation and interaction with downstream signaling molecules, such as GRB2, were not affected, IFNs specifically blocked PDGF- and phorbol ester- but not EGF-stimulated activation of Raf-1, mitogen-activated protein kinases, and tyrosine phosphorylation of an unidentified 34-kDa protein. This inhibition could be detected as early as 5 min after IFN treatments and was insensitive to cycloheximide, indicating that de novo protein synthesis is not required. The IFN-induced inhibition acted upstream of Raf-1 kinase and downstream of diacyl glycerol/phorbol ester, suggesting that protein kinase C (PKC) is the potential primary target. Consistently, downregulation of PKC by chronic phorbol myristate acetate treatment or inhibition of PKC by H7 and staurosporine blocked PDGF- and phorbol myristate acetate- but not EGF-induced signaling and DNA synthesis. Moreover, incubating cells with antisense oligodeoxyribonucleotides of PKC delta eliminated production of PKC delta protein and specifically blocked PDGF- but not EGF-stimulated mitogenesis in these cells. Thus, these studies have elucidated a major difference in the early events of EGF-and PDGF-stimulated signal transduction and, more importantly, revealed a novel mechanism by which IFNs may execute their antiproliferative function.

Interferons block protein kinase C-dependent but not-independent activation of Raf-1 and mitogen-activated protein kinases and mitogenesis in NIH 3T3 cells.

Interferons (IFNs) exert antiproliferative effects on many types of cells. The underlying molecular mechanism, however, is unclear. One possibility is that IFNs block growth factor-induced mitogenic signaling, which involves activation of Ras/Raf-1/MEK/mitogen-activated protein kinase. We have tested this hypothesis by using HER14 cells (NIH 3T3 cell expressing both platelet-derived growth factor [PDGF] and epidermal growth factor [EGF] receptors) as a model system. Our studies showed that IFNs (alpha/beta and gamma) blocked PDGF-and phorbol ester- but not EGF-stimulated DNA synthesis and cell proliferation. While the ligand-stimulated receptor tyrosine phosphorylation and interaction with downstream signaling molecules, such as GRB2, were not affected, IFNs specifically blocked PDGF- and phorbol ester- but not EGF-stimulated activation of Raf-1, mitogen-activated protein kinases, and tyrosine phosphorylation of an unidentified 34-kDa protein. This inhibition could be detected as early as 5 min after IFN treatments and was insensitive to cycloheximide, indicating that de novo protein synthesis is not required. The IFN-induced inhibition acted upstream of Raf-1 kinase and downstream of diacyl glycerol/phorbol ester, suggesting that protein kinase C (PKC) is the potential primary target. Consistently, downregulation of PKC by chronic phorbol myristate acetate treatment or inhibition of PKC by H7 and staurosporine blocked PDGF- and phorbol myristate acetate- but not EGF-induced signaling and DNA synthesis. Moreover, incubating cells with antisense oligodeoxyribonucleotides of PKC delta eliminated production of PKC delta protein and specifically blocked PDGF- but not EGF-stimulated mitogenesis in these cells. Thus, these studies have elucidated a major difference in the early events of EGF-and PDGF-stimulated signal transduction and, more importantly, revealed a novel mechanism by which IFNs may execute their antiproliferative function.

Interactions between inhibitory and excitatory modulatory signals in single submucosal neurons.

Intracellular recordings were made in submucosal neurons from the guinea pig ileum to study the actions of norepinephrine and somatostatin on slow depolarizations induced by 2-chloroadenosine (CADO) and substance P. Local application (by pressure) of CADO and substance P induced a slow depolarization that occurred concomitantly with an increase in input membrane resistance. Norepinephrine, UK-14304 (alpha 2-adrenoceptor agonist), and somatostatin blocked the excitatory responses induced by CADO in a concentration-dependent manner. The alpha 2-adrenoceptor antagonists idazoxan and yohimbine antagonized these inhibitory effects of UK-14304 and norepinephrine. UK-14304 also decreased depolarizations induced by forskolin, but not those induced by the adenosine 3',5'-cyclic monophosphate analogue 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Slow depolarizations induced by substance P were blocked neither by UK-14304 nor by somatostatin. It was previously shown that staurosporine (an inhibitor of various protein kinases) and KT-5720 (an inhibitor of protein kinase A) inhibited slow depolarizations induced by CADO. Here, substance P depolarizations were inhibited by staurosporine and calphostin C (a blocker of protein kinase C) but not by KT-5720. In conclusion, activation of alpha 2-adrenoceptors and somatostatin receptors selectively blocks excitatory responses induced by CADO, most likely by inhibition of adenylyl cyclase and via pertussis toxin-sensitive G proteins. Slow depolarizations induced by substance P are independent of adenylyl cyclase activation and involve activation of protein kinase C.

A role for protein kinases and phosphatases in the Ca 2+-induced enhancement of hippocampal AMPA receptor-mediated synaptic responses

We have investigated the effects of inhibitors of protein kinases and protein phosphatases on the NMDA receptor-independent potentiation of evoked and miniature (m) excitatory postsynaptic currents (EPSCs) induced by the entry of Ca 2+ via voltage-gated Ca 2+ channels in hippocampal CA1 pyramidal neurons. Voltage pulse-induced potentiation was markedly attenuated when evoked in the presence of the protein kinase blockers KN-62, K-252a, or H-7. Bath application of the protein phosphatase inhibitor calyculin A converted the usual transient potentiation of both evoked and spontaneous EPSCs induced by voltage pulses into a more sustained potentiation. Similarly, the introduction of the phosphatase inhibitors microcystin LIZ or okadaic acid into postsynaptic cells, via patch pipettes, also resulted in a sustained increase in the amplitude of mEPSCs. We propose that entry of Ca 2+ into CA1 neurons activates calcium/calmodulin-dependent protein kinase II, which leads to an enhanced responsiveness of synaptic AMPA receptor channels. The enhancement is transient, however, owing to postsynaptic phosphatase activity.

Alpha-melanocyte-stimulating hormone inhibits corticotropin-releasing factor release by blocking protein kinase C.

Cytokine-induced release of corticotropin-releasing factor (CRF) from hypothalamic explants in vitro can be inhibited by femtomolar concentrations of alpha-melanocyte-stimulating hormone (alpha-MSH). Because the mechanism of the anticytokine action of alpha-MSH remains unknown, we examined if the peptide inhibits CRF release by interference with various steps in the activation of CRF release. Previous studies have shown that CRF release is induced by activation of phospholipase A2 (PLA2). Therefore, we examined the effect of alpha-MSH on the action of melittin (MEL), a PLA2 activator. After 60 min preincubation in Krebs-Ringer bicarbonate buffer, medial basal hypothalami were incubated for 30 min with Krebs-Ringer bicarbonate buffer or MEL with or without alpha-MSH (10(-11) to 10(-16) M). CRF release into the incubation medium was measured by RIA. As reported previously none of the alpha-MSH concentrations used changed basal CRF release nor did any concentration of alpha-MSH significantly alter CRF release induced by MEL (10 micrograms/ml). Thus, alpha-MSH alters cytokine-induced CRF release at a step unrelated to the activation of PLA2. Because activation of PLA2 requires an increase in intracellular calcium ion (Ca2+) concentrations, we evaluated the effect of alpha-MSH on the release of CRF induced by a high concentration of potassium (56 mM). This concentration of potassium induced a 3.5-fold increase in CRF release that was not affected by alpha-MSH. Protein kinase C (PKC) stimulates CRF release. Consequently, we examined the effect of alpha-MSH on CRF release induced by phorbol myristate acetate (PMA), which in the presence of Ca2+ stimulates PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

ATPase-promoting dead end inhibitors of the cAMP-dependent protein kinase

The cAMP-dependent protein kinase is a bifunctional enzyme, catalyzing the phosphorylation of the serine and threonine residues in peptides and proteins (kinase activity) as well as the phosphorylation of water (ATPase activity). We have found that several peptides, which serve as inhibitors of the kinase reaction, will either maintain or enhance the ATPase reaction catalyzed by the enzyme. Positively charged dipeptides (e.g. Arg-Arg), as well as small guanidino-containing compounds (e.g. guanethidine) block protein kinase activity yet enhance ATPase activity up to 3.5-fold over that exhibited by the enzyme in the absence of these compounds. In contrast, several nonphosphorylatable peptides, whose primary sequences are based on that of a known substrate (i.e. Leu-Arg-Arg-Ala-Ser-Leu-Gly), such as Leu-Arg-Arg-Ala-Ala-Leu-Gly, Leu-Arg-Arg-Ala-Phe-Leu-Gly, and Leu-Arg-Arg-Ala-Tyr-Leu-Gly, have little or no effect on the rate of the kinase-catalyzed hydrolysis of ATP. An exception to the latter observation is Leu-Arg-Arg-Ala-Cys-Leu-Gly, a cysteine-containing peptide that promotes the protein kinase-catalyzed ATPase reaction by 2.2-fold. We have also found that peptides that possess relatively large amino acid side chain moieties immediately following the arginine dyad (i.e. such as Phe, Tyr, Cys, or Asn at Xaa in Leu-Arg-Arg-Xaa-Ala-Leu-Gly) sharply reduce the rate of enzyme-catalyzed ATP hydrolysis. This suggests that in the presence of peptides containing an -Arg-Arg-Ala- sequence, the enzyme-bound gamma-phosphate of ATP is relatively accessible to water. In contrast, when the latter alanine moiety is replaced by a larger residue, access by water to ATP appears to be hindered. These results indicate that certain structural features associated with the substrate or substrate analog have a profound influence on the manner by which these species interact with the protein kinase. Furthermore, the work described herein demonstrates that it is possible to block the physiologically important kinase reaction and simultaneously promote the energetically wasteful ATPase reaction.

The contributions of protein kinase A and protein kinase C to the actions of 5-HT on the L-type Ca2+ current of the sensory neurons in Aplysia

In the sensory neurons of Aplysia, 5-HT acts through cAMP to reduce current flow through two classes of K+ channels, the S-K + channel and a transient K+ channel (Ikv). In addition, 5-HT increases a voltage-dependent, nifedipine-sensitive Ca2+ current. In this article we show that, while the effect on the S-K+ channel is mediated exclusively by cAMP, the effect on the Ca2+ current can be mimicked by phorbol 12,13-dibutyrate (PDBu) as well as by intracellular injection of cAMP. We then use specific blockers of protein kinase C (PKC) and the cAMP-dependent protein kinase A (PKA) to examine the roles of PKC and PKA in mediating the effect of 5-HT on the nifedipine-sensitive Ca2+ current. We find that H-7, a kinase inhibitor that appears to inhibit PKC more effectively than PKA in intact Aplysia neurons, reverses the increase in the Ca2+ current produced by PDBu. Moreover, H-7 partially blocks the effect of 5-HT on the Ca2+ current without affecting the decrease in the S-K+ current. A more specific PKC inhibitor (the 19-31 pseudosubstrate of PKC) also partially blocks the increase in the Ca2+ current produced by 5-HT, suggesting that this increase is mediated by PKC. Rp-cAMPS, a specific blocker of PKA, did not block the increase in the Ca2+ current produced by 5-HT, suggesting that the effect of 5-HT on this current may be mediated to only a small extent by PKA. The effect of 5-HT on the S-K+ current and the Ca2+ current can also be separated on basis of the time course of their appearance. The fact that the decrease in the S-K+ current precedes the increase in Ca2+ current suggests that there may be a temporal difference in the activation of the two kinase systems.

Mechanism of cAMP-dependent modulation of cardiac sodium channel current kinetics.

beta-Adrenergic modulation is one of the most important regulatory mechanisms of ion channel function. Only recently, however, have beta-adrenergic effects on cardiac Na+ channel activity been recognized, and some diversity of effects has been reported in different preparations. We report studies of protein kinase A-dependent phosphorylation effects on cardiac Na+ current using the macropatch on-cell mode voltage-clamp technique to maintain cytoplasmic composition intact. During the first 5 minutes after addition of 8-(4-chlorophenylthio)cAMP to the bath, the midpoints of both voltage-dependent availability and conductance shifted in the hyperpolarizing direction an average of -7.5 +/- 2.8 mV (n = 31). Moreover, these effects were not species specific; similar results were obtained in canine, rabbit, and guinea pig myocytes, and a similar shift occurred after exposure to 5 microM isoproterenol. Maximum conductance did not change, nor did single-channel conductance. The shifts of conductance and voltage-dependent availability that were induced by protein phosphorylation were distinct from and independent of the slow background shift in kinetics. We measured the background shift to be less than 0.3 mV/min and to be restricted to the channels within the patch. Pretreatment of cells with a blocker of protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H-89), prevented the effect of 8-(4-chlorophenylthio)cAMP while not affecting the background shift in kinetics. Although clearly not the result of addition of a negatively charged phosphate to the inside face of the channel, cAMP-dependent phosphorylation affects the voltage-dependent kinetics, as expected, by an electrostatic interaction with the voltage sensor.

Pre- and postsynaptic actions of nifedipine at an identified cholinergic central synapse of Aplysia.

The effects of the dihydropyridine (DHP) Ca2+ channel antagonist, nifedipine, were studied on the cholinergic synapse between the presynaptic neurones B4/B5 and the postsynaptic neurones B3/B6 located in the buccal ganglion of Aplysia californica. Nifedipine (10 microM) decreased the presynaptic Ca2+ current by 30%-40%. Blockade of DHP-sensitive Ca2+ channels, however, did not affect quantal transmitter release from the presynaptic neurones. Thus, at this synapse, DHP-sensitive Ca2+ channels appear not to be involved in acetylcholine (ACh) release. The postsynaptic response to an ionophoretic application of ACh was decreased by nifedipine, pointing to a blocking action of the drug on the postsynaptic receptor/channel complex. Nifedipine was also found to activate protein kinase C, which in turn induces an increase in the nifedipine-resistant presynaptic Ca2+ influx and in the number of released ACh quanta. These effects of nifedipine could be prevented by a previous application of 1,5-(isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a protein kinase blocker.

Chloride channels in cultured human skeletal muscle are regulated by G proteins.

The regulation of Cl- channels in human myoballs by G proteins was studied using whole-cell and inside-out patch recordings. After perfusion of the cell with 0.1 mM GTP[gamma S], the specific Cl- conductance, GCl, at standard resting potential (-85 mV) was increased from 5.9 microS/cm2 to 103 microS/cm2, and the kinetics upon stepping the potential to positive values was changed from an activating current with very slow inactivation to a fast inactivating current with no potential-dependent activation. These effects were not affected by the simultaneous blockade of several signal cascades involving G proteins. Addition of the protein kinase blockers PKI (25 microM), H8 (10 microM), or of the phospholipase-A2-blocking agent quinacrine (10 microM), had not much influence on these GTP[gamma S] effects. Buffering of the intracellular Ca2+ concentration (0.1 microM) or addition of the Ca2+/calmodulin antagonist trifluoperazine (50 microM) was also without effect. Pre-incubation of the cells with pertussis toxin or with cholera toxin did not change GCl. In excised inside-out patches voltage-clamped at -85 mV, application of GTP[gamma S] influenced the "intermediate" Cl- channel, the Cl- channel type having the highest density in these cells, by increasing the number of transitions in a half-conductance state. The probability of the channel being in one of the two conducting states rose from 0.015 to 0.67, and the kinetics of the single-channel currents was changed so that, on average, it was similar to the whole-cell current kinetics seen after application of GTP[gamma S]. It is concluded that a G protein is directly interacting with these channels.

Effect of parathyroidectomy on serum levels of procollagen type I C-term extension peptide

Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have been shown to relax various types of smooth muscle, e.g. vascular, uterine and gastric. This study demonstrates that PTH and PTHrP both relaxed cholecystokinin octapeptide (CCK)-induced tension in guinea pig gallbladder strips. This relaxation was concentration-dependent. The use of PTHrP (7-34) blocked the relaxant effect of both agents. This suggested PTH and PTHrP were acting through the same receptor. The use of Rp-cAMPs, an inhibitor of cAMP activation of protein kinase A, and H-89, a selective inhibitor of protein kinase A, suggested that cAMP mediated the relaxant action of PTH and PTHrP. The use of iberiotoxin indicated that the high conductance Ca2+-activated potassium channels also mediated the actions of PTH/PTHrP. The use of KT5823, a selective blocker of protein kinase G, also decreased the amount of relaxation induced by PTH/PTHrP. This suggested that crosstalk between the two second messenger (cAMP and cGMP) systems occurred.

The 5-HT4 receptor subtype inhibits K+ current in colliculi neurones via activation of a cyclic AMP-dependent protein kinase.

1. The aim of the present study was to examine the effect of 5-hydroxytryptamine (5-HT) on K+ current in primary culture of mouse colliculi neurones and to identify the 5-HT receptor subtype that could be involved in this effect. 2. The voltage-activated K+ current of the neurones was partially blocked by 8-bromo adenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP). This effect was mimicked by 5-HT and the action of 5-HT could be antagonized by H7, a non specific protein kinase inhibitor, and by PKI, the specific cyclic AMP-dependent protein kinase blocker. 3. A similar cyclic AMP-dependent blockade of the K+ current was found with renzapride (BRL 24,924) and other 5-HT4 receptor agonists such as cisapride, BIMU 8, zacopride and 5-methoxytryptamine (5-MeOT). ICS 205,930, the classical 5-HT4 receptor blocker, could not be used in this study because it inhibited the studied K+ current by itself. However, the novel 5-HT4 receptor antagonist, DAU 6285 blocked the effects of 5-HT and renzapride on the K+ current. 4. The current was insensitive to the 5-HT1 and 5-HT3 receptor agonists (8-hydroxy-2-(di-n-propylamino) tetralin, RU 24,969, carboxamidotryptamine, 2-CH3-5-HT) as well as to 5-HT1, 5-HT2 and 5-HT3 antagonists (methiothepin, ketanserin, ondansetron [GR 38,032]). Moreover, these antagonists did not affect the actions of the tested 5-HT4 receptor agonists. 5. The present results show that part of the voltage-activated K+ current in mouse colliculi neurones is cyclic AMP-sensitive and the blockade of the current by 5-HT involves the 5-HT4 receptor subtype.The putative implication of 5-HT4 receptors in neuronal plasticity, via a blockade of K+ channels, is discussed.