Modulation of delayed rectifier potassium channel by protein kinase C zeta-containing signaling complex in pheochromocytoma cells.

Voltage-dependent delayed rectifier K(+) (Kv) channels are fundamental components in the regulation of neuronal excitability. We found that nerve growth factor (NGF) treatment of PC12 cells induced a hyperpolarizing shift of the Kv current activation curve by about 15 mV. This effect was similar to the effect of the modulatory subunit, Kv beta, on the cloned Kv channel, and required the activity of protein kinase C (PKC)zeta. Since NGF treatment of PC12 cells is known to increase the expression of p62 protein, which binds both to Kv beta and to PKC zeta, our results are consistent with the model in which p62 functions as a physical link in the assembly of signaling complex, PKC zeta-p62-Kv channel. In agreement with this model, the transient expression of p62 induced the same change in the Kv current activation curve as NGF, and the suppression of p62 expression inhibited the effect of NGF. The amount of bound Kv beta to p62 was increased by NGF treatment. These results suggest that the increased p62 protein induces the formation of the signaling complexes, enabling PKC zeta to modulate Kv channels. Thus, this may constitute a new way of modulating Kv channel activities.

Nitric oxide directly activates large conductance Ca2+-activated K+ channels (rSlo).

We investigated whether nitric oxide (NO) directly activates the cloned alpha-subunit of large conductance Ca2+-activated K+ (Maxi-K) channels from rat brain (rSlo), expressed either in HEK293 cells or Xenopus oocytes. In inside-out patches, the application of S-nitroso-N-acetylpenicillamine (SNAP), a NO-releasing compound, reversibly activated the channel shifting the voltage dependent activation curve of the macroscopic Maxi-K current to the left by about 15 mV. Pretreatment of the patches with N-ethylmaleimide to alkylate free sulfhydryl groups did not prevent the effect of SNAP, suggesting that NO may directly interact with the channels. These results suggest that Maxi-K channels might be one of the physiological targets of NO in the brain.

The properties of carbachol-activated nonselective cation channels at the single channel level in guinea pig gastric myocytes.

We investigated the properties of carbachol (CCh)-activated nonselective cation channels (NSC(CCh)) at the single channel level in the gastric myocytes of guinea pigs using a magnified whole-cell mode or an outside-out mode. The channel activity (NPo) recorded in a magnified whole-cell mode increased with depolarization (from -120 to -20 mV) and had the half activation potential of -81 mV under the symmetrical 140 mM Cs+ condition. The single channel conductance depended upon the extracellular monovalent cations with the order of Cs+ (35 pS) > Na+ (25 pS) > Li+ (21 pS). The channel activities markedly diminished or disappeared when external Cs+ was replaced with Na+ or N-methyl-D-glucamate (NMDG+). With Cs+ and Na+ as external cations, the channel showed a monotonic increase in NPo with the increased mole fraction of Cs+ over Na+, and it had an intermediate conductance value in solution containing 67% Cs+ with 33% Na+. These data suggested that the extracellular monovalent cations regulate the whole-cell current of NSC(CCh) by modulating both the open state probability and the unitary conductance, and there is one binding site for the extracellular cations within the pore.

Beta-amyloid peptide induces the expression of voltage dependent outward rectifying K+ channels in rat microglia.

Upregulation of voltage-dependent outward rectifying K+ (Kv) channels has been reported in activated microglia. Since beta-amyloid peptide (A beta) is known to activate microglia, we tested whether the exposure of cultured rat microglia to A beta fragment 25-35 (A beta 25-35) induced the Kv current. A beta 25-35 in 5-200 nM concentration range significantly increased Kv current density, while there was small change in inward rectifying K+ current density. The full length A beta peptide (A beta 1-42) also increased Kv current. However, the control peptide, A beta 35-25, did not induce Kv current. Most of the Kv current induced by A beta was specifically blocked by the presence of antisense deoxyoligonucleotides against Kv1.3, and Kv1.5. Thus, it is concluded that we have identified Kv1.3 and Kv1.5 as the channel types expressed in A beta-treated microglia.

Modulation of large conductance calcium-activated potassium channels from rat hippocampal neurons by glutathione.

We have investigated the modulation of neuronal large conductance Ca2+-activated K+ channels by glutathione. Single channel recordings were made from cultured neonatal rat hippocampal neurons by using excised inside-out patch clamp method. Glutathione, a physiological sulfhydryl specific reducing reagent, increased channel activities in concentration dependent manner with half activation concentration of 710 microM. Conversely, oxidized form of glutathione inhibited channel activities with half inhibition concentration of 520 microM. Our results provide direct evidence that when neuronal large conductance Ca2+-activated K+ channels are exposed to reducing or oxidizing environments, channel activities are increased or decreased in opposite directions due to the redox modification. This may constitute an important regulatory mechanism of neuronal Ca2+-activated K+ channel activities.

Hypotonic swelling increases L-type calcium current in smooth muscle cells of the human stomach.

The purpose of the present study was to characterize the Ca2+ channels in smooth muscle cells from human stomach and to examine the effects of osmotic swelling on the channel activity. Ca2+ channel current with either Ca2+ or Ba2+ as charge carrier was recorded from freshly isolated smooth muscle cells using the conventional whole-cell patch clamp technique. The degree of cell swelling as a result of hypotonic challenge was monitored using a video image analysis system. The changes in intracellular Ca2+ concentration ([Ca2+]i) were measured by microfluorimetry. The pharmacological and voltage activation profile suggests a typical dihydropyridine-sensitive L-type Ca2+ current. Cell swelling, induced by hypotonic challenge, enhanced the amplitude of currents through L-type Ca2+ channels without significant effects on steady-state voltage dependency. After treatment with the L-type Ca2+ channel agonist Bay K 8644 (0.1-2 microM), no further significant increase in calcium channel current or corresponding [Ca2+]i transients were provoked by the swelling. The above results demonstrated that the presence of L-type Ca2+ current in smooth muscle cells of the human stomach and the augmentation of the current are closely associated with the volume increase resulting from hypotonic swelling.

Characterization and regulation of rat microglial Ca(2+) release-activated Ca(2+) (CRAC) channel by protein kinases.

We measured the activity of the Ca(2+) release-activated Ca(2+) (CRAC) channel present in cultured rat microglia, using the whole-cell mode of patch clamp technique. When the concentration of divalent cations in external solution was reduced to the micromolar range, and Ca(2+) chelating agent BAPTA was included in the pipette solution, we were able to record Na(+) current through CRAC channels in single-channel levels. The unitary Na(+) conductance through CRAC channel was 42.5 pS, which was similar to that of Jurkat cell. The Na(+) current activated slowly, reaching the maximal current level in about 10 min after whole-cell patches were made. The time required for the half-maximal activation of the current was 205 s (+/-31), while it was reduced to 84.3 s (+/-17.7) by including IP(3) in the pipette solution as well. The peak currents ranged from 320 to 985 pA, which corresponded to 64-197 channels per cell. We studied the regulation of the current by protein kinase A (PKA) and protein kinase C (PKC). The current was enhanced by the addition of membrane-permeant analogue of cAMP, dibutyryl cAMP. Pretreating cells with PKA inhibitor, H-89, prevented the effect of dibutyryl cAMP. By contrast, the addition of PKC activator, PDBu, reduced the current. Staurosporine, a PKC inhibitor, prevented the effect of PDBu. These results suggest that CRAC channel in rat microglia is under the regulation of PKA and PKC in opposite directions.

Ginsenosides inhibit capsaicin-activated channel in rat sensory neurons.

Ginsenosides isolated from ginseng are biologically active components. In this study, whole-cell and inside-out configurations of patch clamp technique had been used to test the effect of ginsenosides on the capsaicin-activated channels in cultured small diameter sensory neurons of young rat. Ginsenosides (100 microg/ml) decreased the amplitude of capsaicin-activated currents by 78.2% in whole cell mode. Similarly, ginsenosides decreased capsaicin-activated single-channel activities in a dose-dependent manner in inside-out patches. These results indicate that ginsenosides might directly block capsaicin-activated channels, resulting in attenuation of the currents in rat sensory neurons.

Muscarinic receptors controlling the carbachol-activated nonselective cationic current in guinea pig gastric smooth muscle cells.

Muscarinic receptor subtypes controlling the nonselective cationic current in response to carbachol (ICCh) were studied in circular smooth muscle cells of the guinea pig gastric antrum using putative muscarinic agonists and antagonists. Both oxotremorine-M (an M2-selective agonist) and CCh dose-dependently activated the cationic current with EC50 values of 0.21 +/- 0.01 microm and 0.97 +/- 0.06 microM, respectively. In contrast, pilocarpine and McN-A 343 (an M1-selective and a putative M4 agonist) were weak partial agonists. In response to 10/microM CCh, 4-DAMP, methoctramine and pirenzepine dose-dependently inhibited ICCh and had IC50 values of 1.91 +/- 0.2 nM, 0.46 +/- 0.07 microM and 8.33 +/- 0.4 microM, respectively. 4-DAMP, methoctramine and pirenzepine shifted the concentration-response curves of ICCh to the right without significantly reducing the maximal current. Values of the apparent dissociation constant pA2 obtained from Schild plot analysis were 9.24, 7.72 and 6.62 for 4-DAMP, methoctramine and pirenzepine, respectively. Also, pertussis toxin completely blocked ICCh generation. These results suggest that the M2-subtype plays a crucial role in the activation of the ICCh, and a block of the M3-subtype reduces the sensitivity of the M2-mediated response with no significant reduction of maximum response.

Different contractile properties between intralobar and extralobar pulmonary arteries of the rabbit.

In pulmonary circulation, small muscular resistance arteries are known to have different receptor properties and sensitivity to neurotransmitters from those of large elastic conduit arteries. It is, however, not yet certain whether the different properties are primarily due to the diameter or the location of arteries. In the present study, we compared the contractile responses to various agonists among large extralobar (ELPA, diameter: 2-3 mm), large intralobar (ILPA, diameter: 2-3 mm), and small intralobar pulmonary arteries (SPA, diameter: 300-500 microm) of the rabbit. There were no differences in normalized dose-response curves to KCl among three groups. Half maximum doses (EC50 in mM) were 38.0+/-2.0 (n=8, mean+/-SEM) in ELPA, 36.9+/-2.4 (n=10) in ILPA, and 39.0+/-0.9 (n=12) in SPA. Responses to phenylephrine, epinephrine, histamine, serotonin, and PGF2alpha were normalized and expressed as a relative contraction against maximum tension to KCl. In ELPA, the contractile responses to various agents showed the following sequence: KCl>epinephrine>phenylephrine>serotonin>PGF2alpha>histamine. In ILPA and SPA, the sequence was: KCl>histamine>PGF2alpha>serotonin. There was little response to phenylephrine and epinephrine in ILPA and SPA. These results demonstrate that the difference of contractile responses between ELPA and ILPA was more prominent than that between ILPA and SPA, suggesting that the location is more important than the diameter itself in determining the characteristic contractile responses of pulmonary arteries.

beta-adrenergic modulation of maxi-K channels in vascular smooth muscle via Gi through a membrane-delimited pathway.

Direct modulation of large-conductance Ca2+-activated K+ (maxi-K) channel by receptor-associated G protein in rabbit mesenteric arterial smooth muscle cells was studied using the outside-out patch clamp technique. Applying a beta-adrenoceptor agonist (isoproterenol) increased maxi-K channel activity by 75%, and the effect was almost completely abolished by pretreating the cells with pertussis toxin but not with cholera toxin. When the antibody against Gi protein was present in the pipette solution the stimulatory effect of isoproterenol disappeared. These results suggest that beta-adrenoceptor stimulation increases maxi-K channel activity via a membrane-delimited pathway, probably through pertussis toxin-sensitive G protein (Gi).

Identification of a distinct molecular mass G alpha(h) (transglutaminase II) coupled to alpha1-adrenoceptor in mouse heart.

Our previous studies on alpha1-adrenoceptor signaling suggested that G alpha(h) family is a signal mediator in different species. To elucidate the species-specificity of G alpha(h) family in molecular mass, we used the solubilized membranes from mouse heart and the ternary complex preparations containing alpha1-agonist/receptor/G-protein. Binding of [35S]GTPgammaS and the intensity of the [alpha-32P]GTP photoaffinity labeled protein resulting from activation of the alpha1-adrenoceptor were significantly attenuated by the antagonist, phentolamine. The molecular mass of the specific GTP-binding protein was approximately 72-kDa; homologous with G alpha(h) (transglutaminase II) family. Furthermore, immunological cross-reactivity of ternary complex from mouse heart and purified G alpha(h) from rat, guinea pig, and bovine using anti-G alpha(h7) antibody showed that their molecular masses were distinctly different and approximately 72-kDa G alpha(h) from mouse heart was the lowest molecular mass. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the alpha1-adrenoceptor in the ternary complex preparation by anti-G alpha(h7) antibody, the G alpha(h) family protein tightly coupled to alpha1-adrenoceptor. These results demonstrate the species-specificity of G alpha(h) family in molecular mass, especially the lowest molecular mass in mouse.

Nitric oxide directly activates calcium-activated potassium channels from rat brain reconstituted into planar lipid bilayer.

Using the planar lipid bilayer technique, we tested whether NO directly activates calcium-activated potassium (Maxi-K) channels isolated from rat brain. We used streptozotocin (STZ) as NO donor, and the NO release was controlled with light. In the presence of 100-800 microM STZ, the Maxi-K channel activity increased up to 3-fold within several tens of seconds after the light was on, and reversed to the control level several minutes after shutting off the light. Similar activation was observed with other NO donors such as S-nitroso-N-acetylpenicillamine and sodium nitroprusside. The degree of activity increase was dependent upon the initial open probability (P[init]). When the P(init) was lower, the activity increase was greater. These results demonstrate that NO can directly affect the Maxi-K channel activity, and suggest that the Maxi-K channel might be one of the physiological targets of NO in brain.

Modulation of large conductance Ca2+-activated K+ channel by Galphah (transglutaminase II) in the vascular smooth muscle cell.

Among G-proteins, Gh is unique in structural differences in the GTP-binding domain and possessing transglutaminase activity. We have studied the role of G protein in modulation of large conductance Ca2+-activated K+ (Maxi-K+) channel by the inside-out mode of patch clamp in smooth muscle cells from superior mesenteric artery of the rabbit. When the non-hydrolyzable GTP analogue, GTPgammaS, was applied, the channel activity was increased about 2.5-fold. Addition of GDPbetaS resulted in reversal of the GTPgammaS effect. When the Galphah7 antibody was applied, the GTPgammaS-stimulated channel activity was significantly inhibited to control level, suggesting that Galphah is involved in activation of the Maxi-K+ channel in smooth muscle cells.

Effects of ultraviolet light on the tension of isolated human cavernosal smooth muscle from non-diabetic and diabetic impotent men.

The effects of ultraviolet (UV) light (310 nm) on human cavernosal smooth muscles were investigated. Cavernosal strips were obtained from men during penile prosthetic surgery. When the cavernosal strips were irradiated with UV light in an organ bath, after precontraction by norepinephrine (100 nM) for 10, 20, 40 and 90 s at intervals of 3 min, the contracted cavernosal smooth muscles from the impotent men without vascular risk factors (controls) showed relaxation depending on the duration of irradiation. However, the relaxation was not found when the strips were pretreated with methylene blue (10 microM) or their epithelia were denuded. The relaxation response of the cavernosal strips from the patients with diabetogenic impotence was significantly reduced compared with that of the controls. Photorelaxation of human cavernosal strips therefore seems to be dependent on endothelium.

A comparison of the relaxation responses of isolated cavernosal smooth muscles by endothelium-independent and endothelium-dependent vasodilators in diabetic men with impotence.

This study was intended to explore the effects of endothelium-independent, direct smooth muscle relaxants(papaverine, verapamil) and endothelium-dependent vasodilators(acetylcholine, bradykinin, adenosine) on the isolated cavernosal smooth muscle strips taken from diabetic men with impotence. When smooth muscle contraction was evoked with norepinephrine for the study of relaxation to these vasodilators, the tension induced was similar in diabetic and non-diabetic men with importance. Papaverine showed the strongest relaxation response followed by verapamil, acetylcholine, bradykinin and adenosine both in non-diabetic and in diabetic men. Relaxation of the cavernosal tissues to endothelium-independent vasodilators was similar in non-diabetic and diabetic men. However, the relaxation response of the tissues to endothelium-dependent vasodilators was significantly reduced in the diabetic group compared with that in the non-diabetic group (p < 0.05). In conclusion, the impairment of endothelium-mediated relaxation of cavernosal smooth muscle seems to play a more important role in the pathogenesis of diabetogenic impotence rather than the problem of smooth muscle itself. This finding forms a rational basis for the use of intracavernosal injections of vasodilators to induce endothelium-independent relaxation of the cavernosal smooth muscle in the patients with diabetogenic impotence.

Modulation of large conductance calcium-activated K+ channel by membrane-delimited protein kinase and phosphatase activities.

Large conductance Ca(2+)-activated K+ channel was identified and studied in excised inside-out membrane patches of freshly dispersed smooth muscle cells from rabbit gastric antrum. The current-voltage relationship of the single channel was linear from -80 to +80 mV of pipette voltage in which single channel conductance was 249 +/- 17.8 pS (n = 19) in symmetrical concentration of K+ (145 mM) across the patch. Activity of the channel (NPo) depended not only on cytoplasmic calcium concentration but also on membrane potential. MgATP increased NPo in a dose-dependent manner and Mg2+ was prerequisite for the effect. Okadaic acid (100 nM), inhibitor of protein phosphatases, increased NPo further in the presence of MgATP. Therefore, it would be concluded that activity of the calcium-activated K+ channel in gastric smooth muscle cells was controlled by phosphorylation state of the channel protein and the state is further modulated by membrane-delimited protein kinase and protein phosphatase activities.

Sodium-calcium exchange in rabbit heart muscle cells: direct measurement of sarcoplasmic Ca2+ activity.

Calcium ion-selective microelectrodes made with Simon's neutral carrier were used to measure simultaneously sarcoplasmic Ca2+ activity (aiCa) and resting tension (Tr) of rabbit ventricular muscle during reduction and restoration of external sodium ion concentration, [Na]0. Under the same experimental conditions the change in contractile tension (Ta) also measured. In resting muscle the aiCa was 38 +/- 17 nanomolar (mean +/- standard deviation; N = 10). The reduction of [Na]O from 153 to 20 millimolar led to about a threefold increase in aiCa with parallel increases in Tr and Ta. The time course of the change in aiCa was similar to that of the changes in Tr and Ta. The results are consistent with an important role of the sodium-calcium exchange system for regulating sarcoplasmic Ca2+ activity.