Electrophysiological and pharmacological characterization of the K(ATP) channel involved in the K+-current responses to FSH and adenosine in the follicular cells of Xenopus oocyte

The follicular cells surrounding Xenopus oocyte under voltage clamp produce K(+)-current responses to follicle-stimulating hormone (FSH), adenosine (Ade), and intracellularly applied cAMP. We previously reported that these responses are suppressed by the stimulation of P2Y receptor through phosphorylation by PKC presumably of the ATP-sensitive K(+) (K(ATP)) channel. This channel comprises sulfonylurea receptors (SURs) and K(+) ionophores (Kirs) having differential sensitivities to K(+) channel openers (KCOs) depending on the SURs. To characterize the K(+) channels involved in the FSH- and Ade-induced responses, we investigated the effects of various KCOs and SUR blockers on the agonist-induced responses. The applications of PCO400, cromakalim (Cro), and pinacidil, but not diazoxide, produced K(+)-current responses similar to the FSH- and Ade-induced responses in the magnitude order of PCO400 > Cro > pinacidil in favor of SUR2A. The application of glibenclamide, phentolamine, and tolbutamide suppressed all the K(+)-current responses to FSH, Ade, cAMP, and KCOs. Furthermore, both the FSH- and Ade-induced responses were markedly augmented during the KCO-induced responses, or vice versa. The I-V curves for the K(+)-current responses induced by Cro, Ade, and FSH showed outward rectification in normal [K(+)](o), but weak inward rectification in 122 mM [K(+)](o). Also, stimulations of P2Y receptor by UTP or PKC by PDBu markedly depressed the K(+)-current response to KCOs in favor of Kir6.1, as previously observed with the responses to FSH and Ade. These results suggest that the K(+)-current responses to FSH and Ade may be produced by the opening of a novel type of K(ATP) channel comprising SUR2A and Kir6.1.     

Physiological and pharmacological characteristics of quisqualic acid-induced K(+)-current response in the ganglion cells of Aplysia

The extracellular application of either quisqualic acid (QA) or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces an outward current in identified neurons of Aplysia ganglion under voltage clamp. The time course of the QA-induced response is significantly slower than that induced by FMRFamide. The reversal potential for both responses was -92 mV and was shifted 17 mV in a positive direction for a twofold increase in the extracellular K(+) concentration. The QA-induced response was markedly depressed in the presence of Ba(2+), a blocker of inward rectifier K(+)-channel, whereas TEA, a Ca(2+)-activated K(+)-channel (BK(Ca)) blocker, or 4-AP, a transient K(+) (A)-channel blocker, had no effect on the response. The QA-induced K(+)-current was significantly suppressed by CNQX and GYKI52466, antagonists of non-NMDA receptors. However, the application of either kainate or AMPA, agonists for non-NMDA receptors, produced no type of response in the same neurons. The QA-induced K(+)-current response was not depressed at all by an intracellular injection of either guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) or guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS), but the FMRFamide-induced response was markedly blocked by both GDP-betaS and GTP-gammaS in the same cell. Furthermore, the QA- and FMRFamide-induced K(+)-current responses were both decreased markedly when the temperature was lowered to 15 degrees C, from 23 degrees C. These results suggested that the QA-induced K(+)-current response is produced by an activation of a novel type of QA-receptor and that this response is not produced by an activation of the G protein.     

Regulatory roles of Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 2A on the quisqualic acid-induced K+-current response in identified neurons of Aplysia

In identified B6 neurons of Aplysia buccal ganglia under voltage-clamp, application of quisqualic acid (QA) induces a unique slow K(+)-current response independent of G-protein. The response was augmented by raising the temperature in a similar fashion to the Phe-Met-Arg-Phe-NH(2)-induced K(+)-current response mediated by Gi/o. The QA-induced K(+)-current response markedly increased during the perfusion with Ca(2+)-free solution or after the application of W-7, a calmodulin (CaM) inhibitor. It was also enhanced by intracellular application either of H-7, a serine/threonine protein kinase inhibitor, or of KN-93, a Ca(2+)/CaM-dependent kinase II (CaMKII) inhibitor. Furthermore, the QA-induced response was markedly augmented by pre-treatment with 2,3-butanedione monoxime, an inorganic phosphatase. Intracellular application of protein phosphatase 2A (PP2A) significantly augmented the QA-induced response although neither protein phosphatase 1 nor protein phosphatase 2B altered the response. Application of either okadaic acid or calyculin A, protein phosphatase inhibitors, only slightly depressed the QA-induced response. Surprisingly, W-7 had no augmenting effect on the QA-induced response when examined after the application of either okadaic acid or calyculin A. These results suggest that the K(+)-current response is reciprocally but sequentially regulated by PP2A and CaMKII, the response of which the former is facilitating and the latter is inhibiting.     

Sweet taste transduction in hamster: role of protein kinases

Two different second-messenger pathways have been implicated in sweet taste transduction: sugars produce cyclic AMP (cAMP), whereas synthetic sweeteners stimulate production of inositol 1,4, 5-tris-phosphate (IP(3)) and diacylglycerol (DAG). Both sugars and sweeteners depolarize taste cells by blocking the same resting K(+) conductance, but the intermediate steps in the transduction pathways have not been examined. In this study, the loose-patch recording technique was used to examine the role of protein kinases and other downstream regulatory proteins in the two sweet transduction pathways. Bursts of action currents were elicited from approximately 35% of fungiform taste buds in response to sucrose (200 mM) or NC-00274-01 (NC-01, 200 microM), a synthetic sweetener. To determine whether protein kinase C (PKC) plays a role in sweet transduction, taste buds were stimulated with the PKC activator PDBu (10 microM). In all sweet-responsive taste buds tested (n = 11), PDBu elicited burst of action currents. In contrast, PDBu elicited responses in only 4 of 19 sweet-unresponsive taste buds. Inhibition of PKC by bisindolylmaleimide I (0.15 microM) resulted in inhibition of the NC-01 response by approximately 75%, whereas the response to sucrose either increased or remained unchanged. These data suggest that activation of PKC is required for the transduction of synthetic sweeteners. To determine whether protein kinase A (PKA) is required for the transduction of sugars, sweet responses were examined in the presence of the membrane-permeant PKA inhibitor H-89 (10 and 19 microM). Surprisingly, H-89 did not decrease responses to either sucrose or NC-01. Instead, responses to both compounds were increased in the presence of the inhibitor. These data suggest that PKA is not required for the transduction of sugars, but may play a modulatory role in both pathways, such as adaptation of the response. We also examined whether Ca(2+)-calmodulin dependent cAMP phosphodiesterase (CaM-PDE) plays a role in sweet taste transduction, by examining responses to sucrose and synthetic sweeteners in the presence of the CaM-PDE inhibitor W-7 (100 microM). Inhibition resulted in an increase in the response to sucrose, whereas the response to NC-01 remained unchanged. These data suggest that the pathways for sugars and sweeteners are negatively coupled; the Ca(2+) that is released from intracellular stores during stimulation with synthetic sweeteners may inhibit the response to sucrose by activation of CaM-PDE.     

An ATP-regulated and pH-sensitive inwardly rectifying K(+) channel in cultured human proximal tubule cells

Although renal K(+) channels along the nephron have been explored in various animal species, little is known about the K(+) channels in human proximal tubule cells. Using the patch-clamp technique, we investigated the properties of an inwardly rectifying K(+) channel present in the surface membrane of cultured human proximal tubule cells of normal kidney origin. This channel was the most frequently observed K(+) channel in cell-attached patches, and cytoplasmic ATP was required to maintain channel activity in inside-out patches. Its single channel conductance was about 42 pS for inward currents and 7 pS for outward currents under the symmetrical K(+) condition. The ATP effect on channel activity was dose-dependently stimulatory within a range of 0.1 to 10 mM, and a nonhydrolyzable ATP analog, AMP-PNP (3 mM), had no effect on channel activity in either the presence or absence of ATP (1 mM). The channel activity observed in cell-attached patches was reduced to 30 to 50% of controls by a membrane-permeable nonspecific protein kinase inhibitor, K252a (1 microM), or a potent protein kinase A inhibitor, KT5720 (500 nM). In contrast, a membrane-permeable cAMP analog, 8Br-cAMP (100 microM), induced a twofold increase in channel activity. The addition of a catalytic subunit of protein kinase A (PKA-CS, 100 U/ml) to the bath in inside-out patches stimulated channel activity in the presence of 1 mM ATP. Furthermore, the channel activity maintained with 1 mM ATP in inside-out patches was suppressed by internal acidification and enhanced by alkalization. These results suggest that the activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells was ATP-dependent and regulated at least in part by cAMP/PKA-mediated phosphorylation processes and intracellular pH.     

Differential effect of epidermal growth factor on serous and mucous cells in porcine airway submucosal gland

Using a patch-clamp technique, we found that the fresh porcine submucosal gland acinar cells contained two functionally distinct cell populations, i.e. physiologically relevant concentration of acetylcholine (ACh, 30 nM) induced two distinct patterns of electric response in tracheal gland acinar cells. One was characterized by an outstanding oscillatory Cl(-)-current activity, and the other was with poor Cl(-)-current response but with a comparable K(+)-current. We examined the effect of epidermal growth factor (EGF) on the ACh-induced electric responses in these cells. EGF affected only the latter (K(+)-prominent) cell type to potentiate significantly the ACh-induced K(+)-current. An immunohistochemistry revealed that the receptor for EGF was identified preferentially on the mucous, but not serous, cells. Genistein, one of the tyrosine-kinase inhibitors, abolished the augmentation effect of EGF on the ACh-induced current. Thus, we identified the serous cell with a Cl(-)-rich current in response to ACh and the mucous cell with a K(+)-dominant response. Moreover, EGF affected the mucous cells alone to potentiate the ACh-induced electric response. EGF may contribute to the pathophysiological alterations in chronic inflammatory airways both in morphological (mucous cell hypertrophy/hyperplasia) and functional (thick viscous hypersecretion) ways.     

Role of the cAMP sensor Epac as a determinant of KATP channel ATP sensitivity in human pancreatic beta-cells and rat INS-1 cells

Protein kinase A (PKA)-independent actions of adenosine 3',5'-cyclic monophosphate (cAMP) are mediated by Epac, a cAMP sensor expressed in pancreatic beta-cells. Evidence that Epac might mediate the cAMP-dependent inhibition of beta-cell ATP-sensitive K(+) channels (K(ATP)) was provided by one prior study of human beta-cells and a rat insulin-secreting cell line (INS-1 cells) in which it was demonstrated that an Epac-selective cAMP analogue (ESCA) inhibited a sulphonylurea-sensitive K(+) current measured under conditions of whole-cell recording. Using excised patches of plasma membrane derived from human beta-cells and rat INS-1 cells, we now report that 2'-O-Me-cAMP, an ESCA that activates Epac but not PKA, sensitizes single K(ATP) channels to the inhibitory effect of ATP, thereby reducing channel activity. In the presence of 2'-O-Me-cAMP (50 microM), the dose-response relationship describing ATP-dependent inhibition of K(ATP) channel activity (NP(o)) is left-shifted such that the concentration of ATP producing 50% inhibition (IC(50)) is reduced from 22 microM to 1 microM for human beta-cells, and from 14 microM to 4 microM for rat INS-1 cells. Conversely, when patches are exposed to a fixed concentration of ATP (10 microM), the administration of 2'-O-Me-cAMP inhibits channel activity in a dose-dependent and reversible manner (IC(50) 12 microM for both cell types). A cyclic nucleotide phosphodiesterase-resistant ESCA (Sp-8-pCPT-2'-O-Me-cAMPS) also inhibits K(ATP) channel activity, thereby demonstrating that the inhibitory actions of ESCAs reported here are unlikely to arise as a consequence of their hydrolysis to bioactive derivatives of adenosine. On the basis of such findings it is concluded that there exists in human beta-cells and rat INS-1 cells a novel form of ion channel modulation in which the ATP sensitivity of K(ATP) channels is regulated by Epac.     

Protein kinase C suppresses spontaneous, transient, outwards K+ currents through modulation of the Na/Ca exchanger in guinea-pig gastric myocytes

The effect of protein kinase C (PKC) on the Ca2+-activated K+ current (IK,Ca) in guinea-pig gastric myocytes was studied using the whole-cell voltage-clamp technique. At a holding potential of 0 mV, IK,Ca, recorded as spontaneous, transient, outwards currents (STOCs), was markedly inhibited, both in mean amplitude (54 +/- 5%) and frequency (60 +/- 8%) by 1 microM phorbol 12, 13 dibutyrate (PDBu, n = 6). These effects were antagonized by pretreatment with 10 nM bisindolylmaleimide I (n = 5), a selective inhibitor of PKC. The possibility that the inhibition of STOCs was due to direct channel inhibition by PKC was addressed using inside-out or open-cell-attached patch-clamp techniques, the latter established using beta-escin. PDBu did not alter the conductance or open probability of the KCa channel in any mode, suggesting that PKC does not inhibit the KCa channel directly. To study the involvement of the Na/Ca exchanger in the inhibition of STOCs by PDBu, its operation was prevented by replacing Na+ in the internal solution by tris(hydroxymethyl)aminomethane (TRIS) and external Na+ by equimolar K+ and Ca2+-activated inwards K+ currents recorded at a holding potential of 0 mV. Neither the mean amplitude (96 +/- 8%) nor the frequency of these currents was inhibited significantly by 1 microM PDBu (n = 5). Like PDBu, 5 microM 2-(2-[4-(4-nitrobenzyloxy)phenyl]ethyl) isothiourea methanesulphonate (KB-R7943), a selective inhibitor of the reverse mode Na/Ca exchanger, also inhibited the mean amplitude (45 +/- 6%) and frequency (26 +/- 2%) of STOCs at the holding potential of 0 mV (n=6). The results suggest that the suppression of STOCs by PKC is mediated by inhibition of the Na/Ca exchanger.     

Modulation of IK,Ca by phorbol ester-mediated activation of PKC in pleural sensory neurons of Aplysia.

1. The electrophysiological properties of the sensory neurons that mediate withdrawal reflexes in Aplysia are modulated by a number of second messengers. For example, the second messengers adenosine 3',5'-cyclic monophosphate (cAMP) and arachidonic acid modulate the S-K+ current (IK,S) and the calcium-activated K+ current (IK,Ca). Recent evidence suggests that protein kinase C (PKC) may also be an important regulator of cellular plasticity. In the present study we examined the possibility that IK,Ca was modulated by the activation of PKC in the pleural sensory neurons. 2. In voltage-clamped sensory neurons the application of phorbol esters, such as phorbol dibutyrate (PDBu), phorbol myristate (PMA), and phorbol diacetate (PDAc), which activate PKC, caused a dose-dependent increase in a voltage-dependent current with properties that resembled IK,Ca. The inactive isomer of phorbol ester, 4 alpha-phorbol, was without effect. 3. This phorbol ester-sensitive current had the kinetics and pharmacological sensitivity of IK,Ca. The current developed slowly during step depolarizations, showed little inactivation, and was activated at membrane potentials greater than approximately 0 mV. In addition, the current modulated by phorbol esters was blocked by a concentration of tetraethylammonium (TEA) that blocks a component of IK,Ca in the sensory neurons. 4. IK,Ca, which was activated directly by the iontophoretic injection of Ca2+, was also enhanced by PDBu. Moreover, the enhancement of Ca(2+)-elicited responses by PDBu persisted after Ca2+ influx was blocked by cobalt. These results indicate that at least one component of the modulation of IK,Ca by PDBu was independent of the modulation of voltage-dependent Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester-induced inhibition of potassium currents in rat sensory neurons requires voltage-dependent entry of calcium

The whole cell patch-clamp technique was used to examine the effects of protein kinase C (PKC) activation (via the phorbol ester, phorbol 12,13 dibutyrate, PDBu) on the modulation of potassium currents (I(K)) in cultured capsaicin-sensitive neurons isolated from dorsal root ganglia from embryonic rat pups and grown in culture. PDBu, in a concentration- and time-dependent manner, reduced I(K) measured at +60 mV by approximately 30% if the holding potential (V(h)) was -20 or -47 mV but had no effect if V(h) was -80 mV. The PDBu-induced inhibition of I(K) was blocked by pretreatment with the PKC inhibitor bisindolylmaleimide I and I(K) was unaffected by 4-alpha phorbol, indicating that the suppression of I(K) was mediated by PKC. The inhibition of I(K) by 100 nM PDBu at a V(h) of -50 mV was reversed over several minutes if V(h) was changed to -80 mV. In addition, intracellular perfusion with 5 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) or pretreatment with omega-conotoxin GVIA or Cd(2+)-Ringer, but not nifedipine, prevented the PDBu-induced suppression of I(K) at -50 mV, suggesting that a voltage-dependent influx of calcium through N-type calcium channels was necessary for the activation of PKC. The potassium channel blockers tetraethylammonium (TEA, 10 mM) and 4-aminopyridine (4-AP, 3 mM and 30 microM) reduced I(K), but only TEA attenuated the ability of PDBu to further inhibit the current, suggesting that the I(K) modified by PDBu was sensitive to TEA. Interestingly, in the presence of 3 mM or 30 microM 4-AP, 100 nM PDBu inhibited I(K) when V(h) was -80 mV. Thus 4-AP promotes the capacity of PDBu to reduce I(K) at -80 mV. We find that activation of PKC inhibits I(K) in rat sensory neurons and that voltage-dependent calcium entry is necessary for the development and maintenance of this inhibition.     

Analysis of different protein kinase C-dependent events in T cells from allogeneic bone marrow transplantation recipients.

In order to understand the mechanisms underlying the T lymphocyte dysfunction associated to allogeneic bone marrow transplantation (BMT), we assessed two different protein kinase C (PKC) dependent events in T cells from BMT recipients: the PKC-dependent membrane expression and function of the CD69 early activation antigen; and the rapid phorbol ester-induced phosphorylation of PKC protein substrates in lysates from T cells permeabilized with digitonin, in the presence of (gamma-32P)ATP. Most BMT recipient T cells detectably expressed the CD69 surface antigen after 24 h of stimulation with either phorbol 12-myristate 13-acetate (PMA) or anti-CD3 MoAb and PMA, thus indicating that PKC activity is sufficient to induce de novo gene expression. Nevertheless, it is noteworthy that the fluorescent staining intensity with anti-CD69 MoAbs was significantly lower in BMT recipient T cells than in normal T lymphocytes, although no clear-cut correlation was found between the expression of CD69 and the proliferative capacity. However, the pattern of PMA-induced phosphoproteins analysed as early as 1 min after PKC activation in T cells from BMT recipients displaying a low response to mitogenic stimuli, was undistinguishable from that detected in T cells from healthy subjects. In all cases a major 110-kD phosphoprotein was observed, which was inducible with PMA, phorbol 12,13-dibutyrate (PDBu), 1-oleoyl-2-acetylglycerol (OAG) and a phorbol-ester-related activator of PKC (mezerein); moreover, its phosphorylation was blocked by pretreating cells with the PKC inhibitor H-7. Altogether our results suggest that the depressed mitogenic responses, which were also observed in the present study when T cells were stimulated via CD69, cannot be simply attributed to a defective PKC activity.     

Activation of Ca(2+)-dependent K+ channel and Cl- conductance in canine pancreatic acinar cells through a cyclic AMP pathway.

Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca(2+)-dependent K+ channel and Cl- conductance in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp methods. In whole-cell current recordings on isolated cells dialyzed with K(+)-rich solution containing 0.5 mM EGTA, addition of 0.5 mM dibutyryl cAMP (dbcAMP), or 50 microM forskolin to the bath increased outward K+ and inward Cl- currents associated with depolarizing and hyperpolarizing voltage jumps, respectively. In intact cells (cell-attached configurations), addition of 0.5 mM dbcAMP or 50 microM forskolin to the bath increased the opening of single K+ channel. In Ca(2+)-free external solution (bath and pipette) 50 microM forskolin or 0.5 mM dbcAMP application evoked an increase in the opening of single K+ channel in intact cells. Addition of 0.5 mM dbcAMP to the bath solution containing 10 mM EGTA without Ca2+ increased the currents of whole-cell dialyzed with K(+)-rich solution containing 10 mM EGTA. When cell was dialyzed with 20 mM EGTA, dbcAMP, or forskolin application did not increase the whole-cell currents. In excised inside-out patches, addition of the catalytic subunit of cAMP-dependent protein kinase (16 U/ml) in the presence of 0.3 mM ATP to the cytoplasmic face of membrane activated the K+ channel, but 0.1 mM cAMP did not. These results suggest that cAMP-dependent phosphorylation can activate Ca(2+)-dependent K+ channels without increase in intracellular free Ca2+ and cAMP-dependent mechanism can activate Ca(2+)-dependent Cl- conductances without the increase in Ca2+ in canine pancreatic acinar cells.     

Enhancement of potency and efficacy of NADA by PKC-mediated phosphorylation of vanilloid receptor

The search for an endogenous ligand for the vanilloid receptor (VR or TRPV1) has led to the identification of N-arachidonyl dopamine (NADA). This study investigates the role of protein kinase C (PKC)-mediated phosphorylation on NADA-induced membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in dorsal root ganglion (DRG) neurons. In basal state, current induced by 10 microM NADA is 5-10% of the current induced by 1 microM capsaicin or protons at pH 5. However, PKC activator, phorbol 12,13-dibutyrate (PDBu) strongly potentiated ( approximately 15-fold) the NADA-induced current. Repeated application of NADA at short intervals potentiated its own response approximately fivefold in a PKC-dependent manner. PKC inhibitor, bisindolylmaleimide (BIM, 500 nM), a mutant TRPV1 (S800A/S502A), and maximal activation of PKC abolished the potentiation induced by repeated application of NADA. As a further confirmation that NADA could stimulate PKC, pretreatment with NADA potentiated the response of protons at pH 5 (approximately 20 fold), which was dramatically reduced in the mutant TRPV1. In DRG neurons, capsaicin (100 nM) induced a approximately 15 mV depolarization and initiated a train of action potentials compared with 1 microM NADA that produced a approximately 5 mV response. Pretreatment with PDBu induced significantly larger depolarization and potentiated NADA-induced current. Furthermore, exposure of NADA to the intracellular surface of the membrane-induced larger currents suggesting inaccessibility to the intracellular binding site might contribute to its weaker action. These results indicate that NADA is a potent agonist of VR when the receptor is in the PKC-mediated phosphorylation state.     

Stimulation of β-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca²⁺ store depletion these SOCs could also be activated by α-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of β-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective β-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl- sn -glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of β-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.     

Ca(2+)-dependent inhibition of inwardly rectifying K(+) channel in opossum kidney cells

The effect of intracellular Ca(2+) on the activity of the inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS was examined by using the patch-clamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K(+) channel rapidly declined with an application of ionomycin (1 microM) in the presence of 10(-6) M Ca(2+) in cell-attached patches. The application of 10 microM phorbor-12-myristate-acetate (PMA) with 10(-6) M Ca(2+) reduced the K(+) channel activity. Although the channel activity was not influenced by an increase of bath Ca(2+) from 10(-7.5) to 10(-6) M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10(-6) M Ca(2+) in inside-out patches. The inhibitory effect of Ca(2+) with ionomycin on the channel activity was diminished by the pretreatment with a specific PKC inhibitor, GF 109203X (5 microM), in cell-attached patches. By contrast, the application of Ca(2+)/calmodulin kinase II (CaMK II, 300 pM) dramatically increased this channel activity in inside-out patches. In cell-attached patches, the addition of both GF 109203X and cyclospolin A (5 microM), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K(+) channel activity with ionomycin and 10(-6) M Ca(2+). The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 microM) and Ni(2+) (10 microM) to stimulate calcineurin inhibited the channel activity in inside-out patches. Furthermore, the inhibitory effect of PKC or calcineurin on this channel activity was abolished by a removal of Ca(2+) from bath solution. These results suggest that Ca(2+)-dependent inhibitory effect on the inwardly rectifying K(+) channel in OKP cells was mainly mediated by Ca(2+)-PKC-mediated phosphorylation, and that the Ca(2+)-calmodulin-dependent phosphorylation process may be counterbalanced by the Ca(2+)-calmodulin-dependent dephosphorylation process.     

ATP-sensitive potassium channels in rat primary afferent neurons: the effect of neuropathic injury and gabapentin

ATP-sensitive potassium (K(ATP)) currents were examined in dorsal root ganglion neurons from neuropathic and control rats using whole-cell voltage clamp recordings. K(ATP) channel openers (diazoxide and pinacidil) enhanced, and the blocker glibenclamide inhibited an outward current in control neurons in a manner dependent on the pipette ATP concentration. Analysis of reversal potentials showed that this current is carried by K(+) ions. Outward current in cells from rats with peripheral nerve injury was not sensitive to modulators of K(ATP) channels. Gabapentin, a putative K(ATP) channel opener, had minimal effect on currents in either group of neurons. We conclude that normal primary afferent neurons express K(ATP) channels that conduct current which is eliminated by peripheral nerve injury. Gabapentin does not affect this current significantly.     

The small GTP-binding protein RhoA regulates serotonin-induced Na+-current response in the neurons of Aplysia

Application of serotonin (5-HT) induces a slow inward current response in identified neurons of Aplysia ganglia under voltage clamp. The 5-HT-induced current response was depressed in Na+-free media, but augmented in Ca2+-free media, and unaffected by a change in external K+. The 5-HT-induced response was markedly blocked by intracellular injection of guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). After the injection of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), the responses to 5-HT gradually and significantly increased at the initial period, reached its plateau, and finally decreased. Intracellular injection of Clostridium difficile toxin B, a blocker of small G-protein Rho family members such as Rho (RhoA, RhoB and RhoC), Rac and Cdc42, markedly depressed the 5-HT-induced response. Intracellular injection of Clostridium botulinum C3 exoenzyme, a specific blocker of RhoA, RhoB, RhoC, exhibited a similar depressing effect observed with toxin B. In contrast, intracellular injection of recombinant L63RhoA, a constitutively active form of RhoA, significantly augmented the 5-HT-induced response without affecting the resting membrane. These results suggested that the 5-HT-induced Na+-current response might be facilitated by the activation of Aplysia Rho which is closely homologous to RhoA, RhoB or RhoC in mammalian neuron.     

Potassium ion channels operated by receptor stimulation can be activated simply by raising temperature.

Application of either dopamine (DA), acetylcholine (ACh), or histamine (HA) to the identified ganglion cells of Aplysia elicits a K(+)-dependent slow hyperpolarization. When temperature of the bathing solution was raised from 22 to 32 degrees C, these cells were also hyperpolarized with a marked increase in K+ conductance. The warm- and transmitter-induced current responses recorded under voltage clamp were not blocked by either 1 mM Ba2+ or 10 mM TEA. Intracellularly injected guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) depressed both warm- and transmitter-induced K+ responses immediately after the injection. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a gradual, irreversible increase in K+ conductance of the plasma membrane and occluded both responses. Transmitter-induced response markedly decreased when the temperature was raised from 22 to 32 degrees C, suggesting that the response to transmitter was occluded during the warm-induced response. These results suggested that the G-protein regulating the receptor-operated K+ channels could be activated simply by raising temperature.     

Chemical anoxia activates ATP-sensitive and blocks Ca(2+)-dependent K(+) channels in rat dorsal vagal neurons in situ

The contribution of subclasses of K(+) channels to the response of mammalian neurons to anoxia is not yet clear. We investigated the role of ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) currents (small conductance, SK, big conductance, BK) in mediating the effects of chemical anoxia by cyanide, as determined by electrophysiological analysis and fluorometric Ca(2+) measurements in dorsal vagal neurons of rat brainstem slices. The cyanide-evoked persistent outward current was abolished by the K(ATP) channel blocker tolbutamide, but not changed by the SK and BK channel blockers apamin or tetraethylammonium. The K(+) channel blockers also revealed that ongoing activation of K(ATP) and SK channels counteracts a tonic, spike-related rise in intracellular Ca(2+) ([Ca(2+)](i)) under normoxic conditions, but did not modify the rise of [Ca(2+)](i) associated with the cyanide-induced outward current. Cyanide depressed the SK channel-mediated afterhyperpolarizing current without changing the depolarization-induced [Ca(2+)](i) transient, but did not affect spike duration that is determined by BK channels. The afterhyperpolarizing current and the concomitant [Ca(2+)](i) rise were abolished by Ca(2+)-free superfusate that changed neither the cyanide-induced outward current nor the associated [Ca(2+)](i) increase. Intracellular BAPTA for Ca(2+) chelation blocked the afterhyperpolarizing current and the accompanying [Ca(2+)](i) increase, but had no effect on the cyanide-induced outward current although the associated [Ca(2+)](i) increase was noticeably attenuated. Reproducing the cyanide-evoked [Ca(2+)](i) transient with the Ca(2+) pump blocker cyclopiazonic acid did not evoke an outward current.Our results show that anoxia mediates a persistent hyperpolarization due to activation of K(ATP) channels, blocks SK channels and has no effect on BK channels, and that the anoxic rise of [Ca(2+)](i) does not interfere with the activity of these K(+) channels.     

Increased cyclic AMP levels block interleukin 2-induced protein kinase C substrate phosphorylation but not the mitogenic response

Protein kinase C (PKC) has been implicated in the signaling of a number of cellular responses including activation of T cells. In the present report we have evaluated the effect of increased cAMP levels on PKC activation after stimulation of two distinct receptor systems on normal human T cells. PKC substrate phosphorylation can be induced via either the CD3 complex or, to a limited extent, the high affinity interleukin 2 (IL 2) receptor. Substrate phosphorylation via both pathways is shown to be blocked by increased intracellular levels of cAMP. In accordance with previous reports, the CD3-dependent autocrine proliferative response could also be blocked by a cAMP-dependent mechanism. Since direct activation of PKC with a phorbol ester reversed this inhibition, a causal relationship between cAMP-dependent PKC blockage and inhibition of the CD3 response is suggested. In contrast, however, initiation of IL 2-induced proliferation was essentially unaltered by cAMP and could progress in the apparent absence of PKC activity. Thus, this study indicates that IL 2-induced proliferation can under such conditions be completely uncoupled from IL 2-induced PKC activation in normal T cells.