Mechanism of modulation of single sodium channels from skeletal muscle by the β 1-subunit from rat brain

We studied the molecular mechanism of the rat skeletal muscle -subunit (_I) gating kinetics modulation by the brain ₁-subunit by heterologous expression of single sodium channels from _I and ₁ in Xenopus laevis oocytes. Coexpression of ₁ reduced mean open time at –10 mV to 21% when compared to channels expressed by _I alone. Channels formed by _I exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the _I/₁ complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time , was greatly accelerated by ₁, closely resembling mcd of sodium currents from native skeletal muscle. Generally was larger for channels expressed from the pure _I subunit.From our single channel data we conclude that ₁ accelerates the inactivation process of the sodium channel complex.     

On the mechanism of β-adrenergic regulation of the Ca channel in the guinea-pig heart

Dose-response relations for the increase in the amplitude of Ca current (I _Ca) on external application of isoprenaline (ISP) and internally applied cyclic AMP (cAMP) or catalytic subunit of cAMP-dependent protein kinase (C subunit) were established in single ventricular cells of the guinea pig. An intracellular dialysis technique was used. The threshold concentration was for ISP 10^–9 M, for cAMP 3 M (pipette concentration to which 10^–5 M 3-isobutyl-1-methylxanthine was added) and for C subunit around 0.4 M (pipette concentration). The concentrations for the half-maximal effect were 3.7×10^–8 M (ISP), 5.0 M (cAMP) and 0.95 M (C subunit) and for the maximum effect 10^–6 M (ISP), 15–20 M (cAMP) and 3–4 M (C subunit). For all three agents the maximum increase in the Ca current density was similar (a factor of 3–4), suggesting that they converge on the same site of the Ca channel. Accordingly, the effects of cAMP and C subunit onI _Ca were non-additive to those of ISP. From these data the relationship both between concentrations of ISP and cAMP and between those of cAMP and active C subunit in terms of their effects onI _Ca could be estimated and were compared with those obtained in broken cell preparations.A competitive inhibitor of phosphorylation, 5-adenylyl-imidodiphosphate (5 mM), greatly reduced the effects of ISP and C subunit onI _Ca. Cell dialysis with 3 mM adenosine-5-(-thio)-triphosphate, which produces a dephosphorylationresistant phosphorylation, markedly potentiated the effects of ISP and cAMP onI _Ca.The results support the hypothesis that phosphorylation of a protein within, or close to, the Ca channel by cAMP-dependent protein kinase is the mechanism of -adrenergic stimulation.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 38 (membranforschung), Projekt G, and H0579/6-2     

α 1- and β-adrenergic interactions on L-type calcium current in cardiac myocytes

We investigated the mechanism by which ₁-adrenergic activation regulates basal and stimulated whole cell L-type Ca current (I_Ca) in rat ventricular myocytes using the physiological neurotransmitter, norepinephrine (NE, 10M). Stimulation of ₁-adrenoceptors, achieved by NE+10M esmolol (a -receptor antagonist), had no significant effect on basal I_Ca. ₁-adrenergic activation had a marked inhibitory effect on I_Ca elevated by activation (NE+1M) prazosin, an ₁-receptor antagonist) or activation of adenylyl cyclase by forskolin (25M); the inhibitory effect was reversible upon washout. However, ₁-adrenergic stimulation had no significant effect on I_Ca previously increased by intracellular application of cAMP (25M). The inhibitory effect seen on I_Ca elevated by NE showed no significant shift of either I–V or inactivation curves. It is unlikely that the inhibitory effect of ₁-adrenergic stimulation on NE or forskolin-elevated I_Ca is mediated through activation of Ca-dependent protein kinase C or changes in intracellular free Ca (pCa=8.5, EGTA 5 mM) or cAMP-dependent phosphodiesterase. We conclude that ₁-adrenergic inhibition of -adrenergic stimulated-I_Ca is probably mediated through an as yet unknown G-protein. This inhibitory effect could serve as a regulatory feedback mechanism in physiological and pathophysiological settings.     

Interactions of bradykinin,calcium, G-protein and protein kinase in the activation of phospholipase A2 in bovine pulmonary artery endothelial cells

Rise in free cytosolic calcium concentrations [Ca²⁺]_i in response to bradykinin and guanosine 5-O-thiotriphosphate (GTPS) was related to the action of phospholipase A₂ (arachidonic acid release). At 900 M extracellular CaCl₂, bradykinin induced a typical Ca²⁺ movement consisting of an initial [Ca²⁺]_i peak at approximately 400 nM followed by a sustained increase in the steady-state cytosolic Ca²⁺ level at approximately 290 nM. As the extracellular CaCl₂ concentration was reduced to 100 M, the bradykinin induced initial spike was reduced followed by only a marginal increase in steady-state cytosolic Ca²⁺ levels. Treatment of endothelial cells with saponin (0.002% w/w) did not increase [Ca²⁺]_i and saponin treated cells exhibited a very similar pattern of Ca²⁺ mobilization in response to bradykinin. However, with saponin treatment, GTPS (100 M) increased [Ca²⁺]_i at an almost identical tracing exhibited with 50 nM bradykinin stimulation (in either the presence or absence of 0.002% saponin). No additive increase in [Ca²⁺]_i was observed in cells stimulated with both 100 M GTPS and 50 nM bradykinin or in bradykinin stimulated cells subsequently exposed to GTPS. Pertussis toxin (PTX) did not affect the bradykinin induced Ca²⁺ mobilization. However, as we showed previously [1], PTX inhibited bradykinin stimulated arachidonic acid release. These results indicate transduction of the bradykinin signal by G-protein for both phospholipase A₂ (PLA₂) activation and Ca²⁺ mobilization but likely by different G subunits, a PTX sensitive and an insensitive subunit. Furthermore, the bradykinin and GTPS stimulated release of arachidonic acid appears to be only partially dependent on [Ca²⁺]_i. For example, 10 M ionomycin, a calcium ionophore, did not release arachidonic acid at extracellular CaCl₂ concentrations below 300 M while GTPS stimulated a greater release of arachidonic acid at 300 and 100 M CaCl₂ than at 900 M CaCl₂. However, at 100 M CaCl₂, ionomycin increased [Ca²⁺]_i to the same level as bradykinin or GTPS stimulated cells incubated in 900 M CaCl₂.In previously published experiments [1], we showed that phorbol 12-myristate 13-acetate (TPA) augments bradykinin activated arachidonic acid release in endothelial cells. In the absence of bradykinin, TPA had little effect on arachidonic acid release by endothelial cells. However, in the saponin treated cells, TPA alone (in the absence of bradykinin) caused a marked release of arachidonic acid. The bradykinin and TPA activated arachidonic acid releases were additive. The TPA activated release did not require an increase in [Ca²⁺]_i and occurred in the absence of any added extracellular CaCl₂. TPA did not induce an increase in [Ca²⁺]_i in either saponin treated or untreated endothelial cells. This TPA stimulated release of arachidonic acid was totally down-regulated by an 18 h preincubation of the cells in 500 nM TPA but was not inhibited by protein kinase C inhibitor H7.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Role of the GTP-binding protein Gs in the β-adrenergic modulation of cardiac Ca channels

In the heart, the guanosine 5-triphosphate (GTP)-binding protein G_s is activated by hormone binding to -adrenergic receptors and stimulates the intracellular cyclic adenosine 3,5-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A [28]. Additionally, G_s can modulate cardiac Ca channels directly in cell-free systems [57]. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (I _Ca) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained.First, typically, the I _Ca density increased from 12 to 40 A/cm² following application of 1 M isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl₂. However, 1 M ISP enhanced I _Ca only from 9 to 17 A/cm² after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPS (the Rp-isomer of adenosine 3,5-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on I _Ca. Thus, Rpc-AMPS unmasks a GTP-dependent component of the -adrenergic stimulation of I _Ca, which probably reflects the direct stimulation of Ca channels by G_s under block of cAMP-dependent phosphorylation.Second, in cells under dialysis with 100 or 200 M cAMP, bath application of 20–40 M 3-isobutyl-1-methylxanthine (IBMX) enhanced the I _Ca density to about 41 A/cm² indicating saturation of the cAMP pathway. Under this condition, 1 M ISP was without significant effect on I _Ca. This result may suggests that direct G_s stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the G_s pathway in intact myocytes.Third, simultaneous application of 1 M ISP and 40 M IBMX enhanced I _Ca up to densities of around 75 A/cm² during cell dialysis with 100 M cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that G_s is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of G_s may act to prime L-type Ca channels for cAMP-dependent phosphorylation during -adrenergic stimulation of cardiac myocytes.     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.     

Inhibition of the M current in NG108-15 neuroblastoma × glioma hybrid cells

The M current, I _M, a voltage-dependent non-inactivating K current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied inhibition of the M current by bradykinin, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), and methylxanthines. Focal application of 0.1–5 M bradykinin inhibited I _M by about 60%; 5 nM bradykinin inhibited by about 40%. Bath application of 0.1 M and 1 M PDBu diminished I _M to about half of the control value. Staurosporine, a PKC inhibitor, applied for 35–43 min in a concentration of 0.3 M significantly reduced the effect of 1 M PDBu. M current blockage by PDBu could be partly reversed by bath application of H-7 (51–64 M), another PKC inhibitor. These observations suggest that the PDBu effect is really due to activation of PKC. The findings are compatible with the view [Brown DA, Higashida H (1988) J Physiol (Lond) 397:185–207] that the bradykinin effect on I _M is mediated by PKC. However, three further observations suggest that this is only true for part of the bradykinin effect. When the suppression of I _M by 1 M PDBu was fully developed, 0.1 M bradykinin produced a further inhibition of I _M. Down-regulation of PKC by long-term treatment with PDBu reduced the effect of 0.1 M bradykinin significantly but did not abolish it. Staurosporine (0.3 M, applied for 31–46 min) failed to reduce the effect of 5 nM bradykinin significantly. The M current could be reversibly blocked by methylxanthines (caffeine, isobutylmethylxanthine, theophylline) in the millimolar range, probably because of a direct action on the M channels.     

Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current

The effect of tyrosine protein kinase inhibitors on the swelling-induced chloride current (I _Cl-swelling of dog atrial myocytes was studied using the whole-cell patch-clamp recording technique. Currents were measured during hyperpolarizing voltage ramps with potassium currents blocked by cesium. Osmolarity was varied using mannitol. Exposure to hypotonic solution (249 mosmol/kg) activated I _Cl-swelling. Hypertonic solution ( 363mosmol/kg) was used to shrink swollen cells and turn off I _Cl-swelling. In studies on the acute effect of tyrosine protein kinase inhibitors each cell was swollen three separate times. Control, treatment, and washout I _Cl-swelling were compared. Genistein (50–80 M) prevented reactivation of I _Cl-swelling without affecting cell size. The effect of genistein partially subsided upon washout. The effect of genistein on I _Cl-swelling was not mimicked by 80 M daidzein, a related compound that does not inhibit tyrosine protein kinases. When intracellular adenosine 5-0-(3-thiotriphosphate (ATP[S]) was used, genistein did not prevent the reactivation of I _Cl-swelling. Intracellular ATP[S] did not result in a persistent activation of I _Cl-swelling when cell size was returned to control. Acute exposure to 1 M herbimycin A or 100 M tyrphostin 51 did not prohibit the activation of I _Cl-swelling. A 24-h exposure to 1 M herbimycin A did inhibit I _Cl-swelling. The results provide important clues regarding the activation mechanism for I _Cl-swelling and suggest that a tyrosine protein phosphorylation may be necessary, but not sufficient, for activation of I _Cl-swelling.     

Effects of a protein phosphatase inhibitor,okadaic acid,on membrane currents of isolated guinea-pig cardiac myocytes

The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca²⁺ current (I _Ca) was recorded as peak inward current in response to test pulse to O mV. Extracellular application of OA (5–100M) produced an increase ofI _Ca. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline.I _Ca was increased from 11.3±0.8A cm^–2 to 19.0±1.1A cm^–2 (n=4) by 5M-OA in the presence of 1nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the -adrenergic increase ofI _Ca was markedly prolonged by OA. The -adrenergic stimulation of cardiac Ca²⁺ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA onI _Ca is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.     

A dual effect of cardiac glycosides on Ca current in single cells of frog heart

The effects of cardiac glycosides (1 M ouabain, 50 M dihydrooubain, 1 M strophanthidin) on Ca current (I_Ca) were investigated on Csloaded single frog ventricular cells using the wholecell patch-clamp technique (9). Cardiac glycosides exert both inhibitory and stimulatory effects on I_Ca in 20 Cs Ringer solution, but have only a stimulatory effect in O Cs, when the Na,K pump is blocked. The inhibitory response seems related to the inhibition of the Na,K pump by glycosides. The stimulatory effect on I_Ca may contribute to the positive inotropic effect of cardiac glycosides.     

Length-dependent effects of osmotic compression on skinned rabbit psoas muscle fibers

The goal of this study was to characterize the interrelationship between sarcomere length and interfilament spacing in the control of Ca²⁺ sensitivity in skinned rabbit psoas muscle fibers. Measurements were made at sarcomere lengths 2.0, 2.7 and 3.4 m. At 2.7 m the fiber width was reduced by 17% relative to that at 2.0 m and the pCa₅₀ for force development was increased by 0.3 pCa units. In the presence of 5% Dextran T-500 the fiber width at sarcomere length 2.0 m was also decreased by 17% and the Ca²⁺ sensitivity was increased to the same value as at 2.7 m. In contrast, at sarcomere length 2.7 m the addition of as much as 10% Dextran T-500 had no effect on Ca²⁺ sensitivity. At sarcomere length 3.4 m there was an additional 7% compression and the Ca²⁺ sensitivity was increased slightly (0.1 pCa units) relative to that at 2.7 m. However at 3.4 m the addition of 5% Dextran T-500 caused the Ca²⁺ sensitivity to decrease to the level seen at 2.0 m. Given that the skinning process causes a swelling of the filament lattice it is evident that the relationship between sarcomere length and Ca²⁺ sensitivity observed in skinned fibers may not always be applicable to intact fibers. These data are consistent with measurements of Ca²⁺ in intact fibers which indicate that there might be a decline in Ca²⁺ sensitivity at long sarcomere lengths.     

Capsaicin preferentially affects small-diameter acutely isolated rat dorsal root ganglion cell bodies

The effects of capsaicin were tested on 221 acutely isolated dorsal root ganglion neurons of the rat, which ranged in diameter from 15 to 55 m. In a subpopulation of these cells, ranging in diameter from 17.5 to 33 m (n=117), capsaicin (1 M) produced an inward shift in holding current that was associated with an increase in membrane conductance in most cells (114 of 117). These effects of capsaicin were reversible upon washout of the drug. Other cells ranging in diameter from 15 to 52.5 m (n=104) were unaffected in this manner by the 1 m concentration of capsaicin. Capsaicin-sensitive cells had, on average, significantly longer duration action potentials and expressed significantly less I_H than capsaicin-insensitive cells. The relatively long duration action potentials and/or small cell body diameter and paucity of I_H observed in most of the capsaicin-sensitive cells is consistent with their representing C- or A-type sensory neurons.     

Modulation of the inhibitory action of ATP on acetylcholine-activated current by protein phosphorylation in rat sympathetic neurons

Modulation by protein phosphorylation of the relation between acetylcholine (ACh)-activated current (I _ACh) and adenosine triphosphate-(ATP)-activated current (I _ATP) was investigated with the whole-cell voltage-clamp technique in rat sympathetic neurons. During simultaneous activation by 100 M ATP of an inward current, the current evoked by 100 M ACh was reduced to 60–70% of that in the absence of ATP. Effects of compounds that are known to modulate protein phosphorylation were tested by including them in the intracellular solution. The reduction ofI _ACh by ATP was not observed when K252a (1 M), a non-selective protein kinase inhibitor, adenosine 5-O-(3-thiotriphosphate) (ATP[S], 1 mM) or,-methylene ATP (1 mM) were included in the intracellular solution. Activators of protein kinases, adenosine 3,5-cyclic monophosphate (cAMP, 100 M), guanosine 3,5-cyclic monophosphate (cGMP, 100 M), phorbol 12-myristate 13-acetate (PMA, 1 M), also abolished the reduction by ATP ofI _ACh. The effects of okadaic acid, a protein phosphatase inhibitor, were paradoxical: okadaic acid (2 M) itself abolished the reduction by ATP ofI _ACh but it antagonized the abolishment by cAMP or cGMP of the reduction ofI _ACh. Okadaic acid did not affect the disappearance of the reduction ofI _ACh by ATP in the presence of intracellular PMA. The results suggest that the interaction betweenI _ACh andI _ATP is regulated by protein phosphorylation/dephosphorylation. Possible mechanisms underlying the effects of these modulators of protein phosphorylation are discussed.     

The effect of phosphatase inhibitors and agents increasing cyclic-AMP-dependent phosphorylation on calcium channel currents in cultured rat dorsal root ganglion neurones: interaction with the effect of G protein activation

Ca²⁺ channel currents have been recorded in cultured rat dorsal root ganglion neurones. The amplitude of I _Ba(GTPS), recorded in the presence of GTP[S] (200 M) in the patch pipette solution, is enhanced by external application of forskolin (10 M), and there is an increase in the proportion of the rapidly activating component of the current. When forskolin (1 M) is present in the bathing solution at the start of recording, or when 8-bromocyclic AMP (100 M) is present in the patch pipette solution, the amplitude and rate of activation of I _Ba(GTPS) are also increased compared to control I _Ba(GTPS). The effect is mimicked by internal application of a 5 M solution of a phosphopeptide fragment of inhibitor 1 (I1PP), which inhibits phosphatase 1. The enhancement of I _Ba(GTPS) caused by I1PP is not additive with that due to forskolin. Furthermore, the enhancement due to I1PP is reversibly lost when the holding potential is shifted from –80 mV to –30 mV, as was the enhancement due to forskolin and 8-bromocyclic AMP. I1PP also produced a less marked stimulation of the control Ca²⁺ channel current in the absence of G protein activation. The results suggest that phosphorylation regulates the interaction between calcium channels and G proteins in these neurones, and that phosphatase 1 is tonically active to dephosphorylate the relevant protein(s).     

α 2-Adrenoceptors activate dihydropyridine-sensitive calcium channels via Gi-proteins and protein kinase C in rat portal vein myocytes

The presence of functional ₂-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca²⁺]_i) was estimated using emission from the dye Fura-2. Activation of ₂-adrenoceptors by clonidine (an ₂-adrenoceptor agonist) or noradrenaline (a non-selective -adrenoceptor agonist), both in the presence of 0.1 M prazosin to block ₁-adrenoceptors, caused a slow and sustained increase in [Ca²⁺]_i which was inhibited by 0.1 M rauwolscine (an ₂-adrenoceptor antagonist). A similar Ca²⁺ response was obtained with oxymetazoline (a selective _2A-adrenoceptor agonist) suggesting that the increase in [Ca²⁺]_i resulted from activation of the _2A-adrenoceptor subtype. The increase in [Ca²⁺]_i did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The _2A-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. In addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 M prazosin. Taken together, these results indicate that _2A-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Wholecell patch-clamp experiments showed that _2A-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels. Concomitant with calcium influx, _2A-adrenoceptor activation induced a 10- to 15-mV depolarization. Similar effects on both calcium channel current and [Ca²⁺]_i were obtained with mastoparan, an activator of Gi-proteins. Activation of calcium influx by both _2A-adrenoceptors and mastoparan was reduced by treatment with pertussis toxin and GF 109203X (a protein kinase C inhibitor). These data suggest that activation of protein kinase C through a transduction pathway involving Gi-proteins phosphorylates voltage-activated L-type calcium channels and thus, increases their opening probability.     

Distribution of the isoprenaline-induced chloride current in rabbit heart

Current density of the isoprenaline-induced chloride current (I _Cl) was measured in sino-atrial (SA) node cells and atrial and ventricular myocytes dissected enzymatically from the rabbit heart. In addition to the conventional voltage clamp method the whole-cell patch clamp method using nystatin was employed to avoid run-down of I _Cl in dialysed cells. Isoprenaline (0.3 M) failed to induce I _Cl in the 20 atrial cells examined. The integrity of the -adrenergic system was established by recording the response of the Ca²⁺ current in the same cell. Both isoprenaline and acetylcholine failed to affect the background membrane conductance in the 20 SA node cells studied. Myocytes isolated from the epicardial region of the left ventricular wall showed relatively higher I _Cl density (24.9±12.1 S/F) than those from the endocardial side (12.3±8.5 S/F). We conclude that -receptor-operated I _Cl is insignificant in atrial and SA node cells.     

α-Adrenergic activation of the muscarinic K+ channel is mediated by arachidonic acid metabolites

Phenylephrine (10 M), added in the bathing solution, stimulated the cardiac muscarinic K⁺ channel (I_K.ACh) in the cell-attached patch. The pipette solution contained 10 M atropine and 100 M theophylline to block the muscarinic acetylcholine and adenosine receptors, respectively. The channel activation induced by phenylephrine was blocked by prazosin, an ₁-antagonist, indicating that ₁-adrenergic receptor mediates the response. Phenylephrine-induced activation was prevented by nordihydroguaiaretic acid, a lipoxygenase inhibitor, and AA-861, a 5-lipoxygenase inhibitor, but was not affected by indomethacin, a cyclooxygenase inhibitor. These observations suggest that 5-lipoxygenase metabolites of arachidonic acid may be involved in the -adrenergic activation of I_K.ACh.On leave from Kyowa Hakko Kogyo Co., Pharmaceutical Research Laboratories; Nagaizumi-cho, Sunto-gun, Shizuoka 411, Japan     

Soluble β2-μ-Associated and β2-μ-Free HLA Class I Heavy Chain Serum Levels in Interferon-α Nonresponder Chronic Hepatitis C Patients. Markers of Immune Activation, and Response to Antiviral Retreatment

The serum levels of soluble ₂--associated and ₂--free HLA class I heavy chains were determined in 28 interferon- nonresponder chronic hepatitis C patients retreated with interferon- plus ribavirin and in 70 healthy subjects. The baseline levels of ₂--associated and ₂--free HLA class I heavy chains were significantly higher in patients than in healthy controls(P = 0.001). The levels of ₂--associated HLA class I heavy chains significantly increased in responder patients with respect to nonresponders at the third month of treatment(P = 0.03). At the sixth month of treatment and after 6 months of follow up the levels of ₂--associated HLA class I heavy chains decreased in responder patients and increased in nonresponders. The levels of ₂--free HLA class I heavy chains showed only minor changes during and after treatment. We suggest that the determination of hepatitis C virus RNA levels combined with soluble ₂--associated HLA class I heavy chains, as a marker of immune activation, could identify interferon- non responder chronic hepatitis C patients most likely to respond to a retreatment with interferon- plus ribavirin.