Mechanism of hyperthyroidism-induced modulation of the L-type Ca2+ current in guinea pig ventricular myocytes

The positive inotropic effects of thyroid hormone in the heart, increased force and velocity of contraction have been mostly attributed to modulation of myosin ATPase isoenzymes (V₁, V₂ and V₃), and sarcoplasmic reticulum Ca²⁺ pumping activity. In addition, we have suggested that the effects on ventricular contraction result from a thyroid hormone-induced increase in L-type Ca²⁺ current (I _{Ca, L}). Due to the central role of I _{Ca, L} in excitation-contraction coupling, we studied mechanisms whereby thyroid hormone augments this current. Since thyroid hormone modulates adenylate cyclase activity in various tissues, we tested the hypothesis that the hormone activates adenylate cyclase, leading to increased cyclic adenosine monophosphate (cAMP) levels, protein kinase A activation, Ca²⁺ channel phosphorylation and increased I _{Ca, L}. We therefore stimulated or inhibited different sites along the adenylate cyclase cascade, and measured I _{Ca, L} and isometric twitch in ventricular myocytes and papillary muscles from euthyroid and hyperthyroid guinea pigs. Our major findings were as follows. In euthyroid myocytes, 0.1 M isoproterenol (Iso) increased I _{Ca, L} (at V _M=0 mV) from –7.04±0.72 to –22.26±1.88 pA/pF, P<0.05, while in hyperthyroid myocytes (I _{Ca, L}=-21.48±2.94 pA/pF), Iso was ineffective. In euthyroid myocytes, intracellular application of cAMP (50 M) was as potent as Iso, but ineffective in hyperthyroid myocytes. In hyperthyroid myocytes, a protein kinase A inhibitor (2 M) lowered I _{Ca, L} from –26.82±1.54 to -10.17±1.70 pApF (P<0.05), but had no effect in euthyroid myocytes. In hyperthyroid myocytes, acetylcholine (ACh) (1 M) decreased I _{Ca, L} from –26.86±1.49 to –18.33±1.25 pA/pF (P<0.05), while in euthyroid myocytes ACh decreased I_{Ca, L} from –6.80±0.61 to –6.00±0.39 pA/pF (NS). Accordingly, in hyperthyroid papillary muscles, ACh decreased twitch tension by 36.4±2.8%, but in euthyroid preparations only by 9.4±5.1% (P<0.05). These findings suggest that thyroid-hormone-induced increase in I _{Ca, L} contributing to positive inotropy, is mediated by activation of the adenylate cyclase cascade.     

Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes

The mechanism of the action of acetylcholine (ACh) on the L-type calcium current (I _Ca,L) was examined using a whole-cell voltage-clamp technique in single sino-atrial myocytes from the rabbit heart. ACh depressed basal I _Ca,L at concentrations in the range 0.05–10 M, without previous -adrenergic stimulation. The ACh-induced reduction of I _Ca,L was reversed by addition of atropine, indicating that muscarinic receptors mediate it. Incubation of cells with a solution containing pertussis toxin led to abolition of the ACh effect, suggesting that this effect is mediated by G proteins activated by muscarinic receptors. Dialysis of cells with protein kinase inhibitor or 5-adenylyl imidodiphosphate, inhibitors of the cAMP-dependent protein kinase, decreased basal I _Ca,L by about 85% and suppressed the effect of ACh. The ACh effect was also absent in cells dialysed with a non-hydrolysable analogue of cAMP, 8-bromo-cAMP. The results suggest that, in basal conditions, a large part of the L-type calcium channels should be phosphorylated by protein kinase A stimulated by a high cAMP level correlated with a high adenylate cyclase activity. The depressing effect of ACh on I _Ca,L may occur via inhibition of the high basal adenylate cyclase activity leading to a decrease of cAMP-dependent protein kinase stimulation and thus to a dephosphorylation of calcium channels.     

Effects of calcitonin gene-related peptide on membrane currents in mammalian cardiac myocytes

We examined the effects of calcitonin gene-related peptide (CGRP) on the membrane currents of single atrial and ventricular cells of guinea pig heart. The tightseal whole-cell voltage-clamp technique was used. In atrial cells, like isoproterenol, CGRP increased the L-type Ca channel current (I _Ca.L) in a concentration-dependent manner. Human CGRP-(8-37), a putative CGRP receptor antagonist, completely abolished the CGRP-induced increase of I _Ca.L. Although the effects of CRGP were similar to those of isoproterenol, propranolol, a -adrenergic receptor antagonist, did not affect the CGRP-induced increase of I _Ca.L. After I _Ca.L had been maximally activated by isoproterenol (2 M) or intracellular cyclic adenosine 5-monophosphate (100 M), CGRP failed to increase I _Ca.L. Acetylcholine antagonized the effects of CGRP on I _Ca.L. Unlike the effects on atrial cells, CGRP had no significant effects on the membrane currents of ventricular myocytes. Thes results indicate that CGRP increases I _Ca.L via adenylate cyclase activation by binding to specific membrane receptors in cardiac atrial myocytes. Furthermore, CGRP receptors are expressed in atrial cells but probably not in ventricular cells.     

The activation of Ca2+-dependent K+ conductance by adrenaline in mouse peritoneal macrophages

Responses to adrenaline in mouse peritoneal macrophages were investigated with perforated and cell-attached patch-clamp recording, and with a combination of the perforated-patch recording and fura-2 fluorescence measurements. Extracellularly applied adrenaline induced a transient outward current (4–10s in duration, 100–500 pA in amplitude) at –40 mV associated with a marked increase in conductance. The adrenaline-induced current [I _o (Adr)] reversed polarity near –80 mV. The reversal potential depended distinctly on the external K⁺ concentration but not on external Cl^– concentration. Removal of external Ca²⁺ did not affect I ^o(Adr) within 2–4 min but subsequent responses to adrenaline were progressively depressed. In contrast, treatment with an intracellular Ca²⁺ chelator, the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid completely abolished I _o(Adr). Furthermore, I _o(Adr) was blocked by bath-applied quinidine and charybdotoxin, but not by tetraethylammonium or apamin. Extracellular application of an ₁-adrenoceptor agonist phenylephrine and of noradrenaline mimicked I _o(Adr). On the other hand, I _o(Adr) was antagonized by a non-selective -adrenoceptor antagonist phentolamine (0.2 M) and an ₁-adrenoceptor antagonist prazosin (0.2 M), but was not affected by an ₂-adrenoceptor antagonist yohimbine (1 M) or a -adrenoceptor antagonist propranolol (1 M). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed the activation of single-channel currents with a conductance of 40 pS during application of adrenaline outside the patch. Parallel measurements of membrane current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca²⁺]_i and an increase in K⁺ conductance. Therefore, it is concluded that adrenaline activates a Ca²⁺-dependent K⁺ conductance by release of Ca²⁺ from internal stores through an activation of an ₁-adrenoceptor.     

Inhibitory effects ofγ-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

The effects of mouse recombinant-interferon (-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg⁹-bradykinin and prostaglandin E₂ (PGE₂) have been studied using cultures of neonatal calvarial bones and analyzing the release of⁴⁵Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects of-IFN and indomethacin on formation of PGE₂ in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mol/l) totally abolished bradykinin (1 mol/l) induced⁴⁵Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE₂ (1 mol/l).-IFN (1000 U/ml) almost totally inhibited⁴⁵Ca release stimulated by bradykinin (1 mol/l), but the inhibitory effect could only be partially overcome by PGE₂.-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg⁹-bradykinin (1 mol/l) on⁴⁵Ca release. The stimulatory effects of PGE₂ (1 mol/l) on radioactive calcium mobilization was partially inhibited by-IFN (1000 U/ml), whereas indomethacin (1 mol/l) was without effect. The inhibitory effect of-IFN on⁴⁵Ca release stimulated by bradykinin and PGE₂ was dose-dependent with threshold for action at 3–30 U/ml. Comparative dose-response curves showed that-IFN was most potent as inhibitor of bradykinin induced⁴⁵Ca release. Bradykinin (1 mol/l) significantly stimulated PGE₂ formation by a mechanism that was completely inhibited by indomethacin (1 mol/l).-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE₂ formation. These data show that i)-IFN is a potent inhibitor of bone resorption induced by bradykinin and bradykinin analogues and ii) that the mechanism of action can be mainly explained by an inhibition of kinin induced prostaglandin biosynthesis. The results, however, also show that-IFN can inhibit bone resorption by mechanisms unrelated to prostaglandin formation.     

Voltage-clamp studies of the Na+/glucose cotransporter cloned from rabbit small intestine

Inward Na⁺ currents associated with the cloned intestinal Na⁺/glucose cotransporter expressed in Xenopus oocytes have been studied using the two-microelectrode voltage-clamp method. The steady-state current/voltage relations showed voltage-dependent (V _m from +20 to –75 mV) and relatively voltage-independent (V _m from –75 to –150 mV) regions. The apparent I _max for Na⁺ and glucose increased with negative membrane potentials, and the apparent K _0.5 for glucose (K _0.5 ^Glc ) depended on V _m and [Na]_o. Increasing [Na]_o from 7 to 110 mmol/l had the same effect in decreasing K _0.5 ^Glc from 0.44 to 0.03 mmol/l as increasing the V _m from –40 to –150 mV. The I/V curves under saturating conditions (20 mmol/l external sugars and 110 mmol/l [Na]_o) were identical for d-glucose, d-galactose, -methyl d-glucopyranoside and 3-O-methyl d-glucoside. The specificity of the cotransporter for sugars was: d-glucose, d-galactose, -methyl d-glucopyranoside > 3-O-methyl d-glucoside d-xylose > d-allose d-mannose. K _i for phlorizin ( 10 mol/l) was independent of V _m at saturating [Na]_o. We conclude that a variety of sugars are transported by the cloned Na⁺/glucose cotransporter at the same maximal rate and that membrane potential affects both the maximal current and the apparent K _0.5 of the cotransporter for Na⁺ and glucose.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Potentiating and depressant effects of metabotropic glutamate receptor agonists on high-voltage-activated calcium currents in cultured retinal ganglion neurons from postnatal mice

This study was aimed at clarifying the role of metabotropic glutamate receptors (mGluRs) in the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in postnatal mouse retinal ganglion neurons (RGNs). RGNs were maintained for 1–2 weeks in vitro by adding brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) to the culture medium. In order to select these cells for electrophysiological measurements, RGNs were vitally labelled with an antibody against Thy-1.2. Voltage-activated Ca²⁺ currents [I _Ca(V)] were recorded with patch electrodes in the wholecell configuration. It was found that racemic ±-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD) or its active enantiomer 1S,3R-ACPD rapidly and reversibly either enhanced or depressed I _Ca(V). Quisqualate (QA), l-2-amino-4-phosphonobutyrate (l-AP4) and the endogenous transmitter glutamate induced similar effects when ionotropic glutamate receptors were blocked with d-2-amino-5-phosphonovalerate (d-APV) and 6,7-dinitroquinoxaline-2, 3-dione (DNQX). - Conotoxin GVIA (-CgTx GVIA), but not nifedipine prevented modulation of I _Ca(V) by mGluR agonists. The depression of I _Ca(V) by t-ACPD was irreversible when cells were dialysed with guanosine-5-O-(3-thiotriphosphate) (GTP[-S]). Ratio measurements of fura-2 fluorescence in Thy-1+ cells showed that neither t-ACPD, QA nor l-AP4 affected [Ca²⁺]_i by liberation of Ca²⁺ from intracellular stores. Our results suggest that cultured RGNs express mGluRs. These receptors cannot induce Ca²⁺ release from intracellular stores but regulate [Ca²⁺]_i by a fast and reversible, G-protein-mediated action on a subpopulation of voltage-activated Ca²⁺ channels.     

Inositol 1,4,5-trisphosphate mediates adrenaline activation of K+ conductance in mouse peritoneal macrophages

In mouse peritoneal macrophages, ₁-adrenoceptor stimulation evokes a Ca²⁺-dependent K⁺ current [I ₀(Adr)] [Hara et al. (1991) Pflügers Arch 419:371–379]. The roles of D-myo-inositol 1,4,5-trisphosphate (InsP ₃) and a GTP-binding protein (G protein) in I ₀(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of lnsP ₃ (5–50 M) evoked transient outward currents [I ₀(InsP ₃)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5-[3-thio]triphosphate (GTP[S], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10–90 s. I ₀(InsP ₃) and the GTP[S]-induced current {I ₀(GTP[S])} were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K⁺ concentration. Fura-2 measurements revealed that I ₀(InsP ₃) and I ₀(GTP[S]) result from a rise in intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Removal of extracellular Ca²⁺ did not abolish I ₀(InsP ₃) and I ₀(GTP[S]). Both were blocked by bath-applied charybdotoxin. Intracellular D- myo-inositol 1,3,4,5-tetrakisphosphate (InsP ₄, 50 M) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP ₃(2,4,5), 20 M] elicited an outward current at -40 mV. I₀(InsP ₃) was completely blocked by prior dialysis with the InsP ₃ receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5-[2-thio] diphosphate (GDP[S], 2 mM) or heparin (5 mg/ml) together with GTP[S] in the patch pipette solution completely blocked I ₀(GTP[S]). These results indicate that intracellular injection of InsP ₃ or GTP[S] mimic I ₀(Adr). Furthermore, intracellular dialysis with heparin (3 mg/ ml) or GDP[S] (2 mM) greatly accelerated a run-down of I ₀(Adr). On the other hand, I ₀(Adr) was markedly prolonged in a cell dialysed with GTP[S] (0.2 mM). Therefore, it is concluded that I ₀(Adr) results from stimulation of ₁-adrenoceptor and InsP ₃ formation via a G protein.     

Protein kinase C as mediator of arachidonic acid-induced decrease of neuronal M current

The M current, I _M, of NG108-15 neuroblastoma×glioma hybrid cells, a non-inactivating K⁺ current, is decreased by arachidonic acid (5–25 M), often after an initial transitory increase. To test the possibility that the decrease is caused by activation of protein kinase C (PKC) we used the PKC 19–31 peptide, which is an effective inhibitor of PKC. With 1 M peptide in the pipette solution the normally observed strong reduction of I _M by 1 M phorbol 12,13-dibutyrate (PDB) was almost totally prevented, indicating that PKC is completely inhibited; also the voltage dependence of the M conductance, g _M(V), was shifted to more negative membrane potentials. In the presence of 1 M peptide the effect of 25 M arachidonic acid on I _M was significantly reduced, suggesting that the effect, or at least a large part of it, is mediated by PKC.     

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.     

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.     

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.     

Die optische Rotationsdispersion isolierter Paraproteine

Zusammenfassung Die aus dem optischen Drehungsvermögen abgeleiteten Konstanten elektrophoretisch isolierterA-Paraproteine werden mitgeteilt. Die Dispersionskonstante _c weist keine Unterschiede zwischen den 3 ParaproteingruppenG,A undM auf. Der nach dem Verfahren vonMoffitt undYang ermittelte Parameterb ₀ wurde zu Schätzung des-Helixgehaltes benutzt. Er betrug in den 7 untersuchten Paraproteinen 0. Für den Parameter —a ₀ ergab sich ein Mittelwert von 276,0±35,1. FürG-Paraprotein wurde in früheren Untersuchungen ein solcher von 312,8±20,8, fürM-Paraprotein 217,9±26,7 gefunden. Der Mittelwertsvergleich zeigte Signifikanz der Konstantea ₀ für jede der 3 Paraproteingruppen.a ₀ beschreibt demnach gruppenspezifische Eigenschaften von Paraproteinen. Die für den Wert vona ₀ maßgeblichen strukturellen Voraussetzungen sind kaum bekannt. Sie werden am ehesten die die spezifischen Antigendeterminanten tragenden H-Ketten des Paraproteinmoleküls betreffen.

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Summary The constants of the optical rotatory dispersion of electrophoretically isolatedA-paraproteins are communicated. There is no difference between theG,A andM-paraprotein group with respect to the dispersion constant _c . The parameterb ₀ was measured according toMoffitt andYang. The-Helix-content calculated fromb ₀ of 7A-paraproteins was sero (0).The mean value of the parameter —a ₀ was 276±35,1. In earlier experiments it was found that —a ₀ forG-paraproteins is 312,8±20,8 and forM-paraproteins 217,9±26,7. The parametera ₀ of each group differs significantly from the others; in other words,a ₀ is group specific. The structural implications of these findings are discussed.

     

Isoproterenol modulates the calcium channels through two different mechanisms in smooth-muscle cells from rabbit portal vein

Previous data from our laboratory indicated that the slow Ca²⁺ channel of vascular smooth muscle cells was regulated by cyclic nucleotides. In the present study, the effects of isoproterenol (ISO) on L-type calcium current (I _Ca(L)) were investigated in freshly-isolated single smooth-muscle cells from the rabbit portal vein using the whole-cell voltage-clamp technique. With high-Cs⁺ solution in the pipette and physiolocial salt solution (containing 2.0 mM Ca²⁺) in the bath, (I _Ca(L)) was recorded. At a holding potential of –80 mV, low concentrations of ISO ( 100 nM) increased I _Ca, whereas higher concentrations (1–100 M) transiently increased I _Ca but then inhibited it persistently. At 10 M ISO, I _Ca was initially increased by 44±9%, and was subsequently decreased by 24±3%. Pretreatment of cells with 30 M H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride] caused the first phase to persist and the second inhibitory phase to disappear. Intracellular application of 1 mM GDP[S] (guanosine 5-O-2-thiodiphosphate) abolished both phases of ISO action. In contrast, intracellular application of 100 M GTP caused the initial stimulatory phase of ISO action to be significantly potentiated; the later inhibitory phase was slightly diminished. In addition, the activated G protein subunit (G_s ) mimicked the stimulatory effect of ISO. Pertussis toxin had no effect on either phase of the ISO action. These results suggest that ISO modulates the Ca²⁺ channel through mechanisms that involve the pertussis-toxin-insensitive G protein(s). That H-7, a nonspecific inhibitor of protein kinases, blocked the second phase but not the first phase indicates that the actions of ISO are mediated via two different pathways. One pathway (for inhibition) is more indirect, and may be mediated by the adenylate cyclase/cAMP/protein-kinase-A cascade. The other pathway (for stimulation) is more direct, and may reflect a type of G protein gating of the Ca²⁺ channel.     

Modulation of Ca2+ oscillation and apamin-sensitive,Ca2+-activated K+ current in rat gonadotropes

In rat pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) stimulates rhythmic release of Ca²⁺ from stores sensitive to inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃ ], which in turn induces an oscillatory activation of apamin-sensitive Ca²⁺-activated K⁺ current, I _K(Ca). Since GnRH also activates protein kinase C (PKC), we investigate the action of PKC while simultaneously measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) and I _K(Ca). Stimulation of PKC by application of phorbol 12-myristate 13-acetate (PMA) did not affect basal [Ca²⁺]_i. However, PMA or phorbol 12,13-dibutyrate (PdBu), but not the inactive 4-phorbol 12,13-didecanoate (4-PDD), reduced the frequency of GnRH-induced [Ca²⁺]_i oscillation and augmented the I _K(Ca) induced by any given level of [Ca²⁺]_i. The slowing of oscillations and the enhancement of I _K(Ca) were mimicked by synthetic diacylglycerol (1,2-dioctanoyl-sn-glycerol) and could be induced during ongoing oscillations that had been initiated irreversibly in cells loaded with guanosine 5-O-(3-thio-triphosphate) (GTP-[S]). In contrast, when oscillations were initiated by loading cells with Ins(1,4,5)P ₃, phorbol esters enhanced I _K(Ca) without affecting the frequency of oscillation. The protein kinase inhibitor, staurosporine, reduced I _K(Ca) without affecting [Ca²⁺]_i and partially reversed the phorbol-ester-induced slowing of oscillation. Therefore, activation of PKC has two rapid effects on gonadotropes. It slows [Ca²⁺]_i oscillations probably by actions on phospholipase C, and it enhances I _K(Ca) probably by a direct action on the channels.     

Detection and quantitation of cell-surface sugar receptor(s) ofLeishmania donovani by application of neoglycoenzymes

Promastigote culture forms of the log growth phase ofLeishamania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety, was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of -galactosidase fromEscherichia coli: -d-lactose, -d-thiogalactose, -d-mannose, -l-rhamnose, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylglucosamine, the - and -glucosides maltose and cellobiose, -d-xylose, -d-mannose-6-phosphate, the -galactoside melibiose, -l-fucose, and -d-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigoteLeishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, andN-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealedK _D values of around 100mm and around 10⁴ binding sites for the polyvalent ligands.     

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.     

ω-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca²⁺]_i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca²⁺]_i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. -Conotoxin GVIA (-CgTx, 100 nM) inhibited action-potential-mediated Ca²⁺ influx by 79%, while nitrendipine (1 M) had little effect. -Grammotoxin SIA (-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca²⁺ influx as effectively as did -CgTx, suggesting that -GsTx blocks N-type Ca²⁺ channels. In contrast to block by -CgTx, the block produced by -GsTx reversed upon washout of the peptide. -GsTx (270 nM) blocked 80%, and -CgTx (1 M) blocked 64%, of whole-cell Ca²⁺ current (I _Ca) elicited by step depolarization to 0 mV from a holding potential of –80 mV. -GsTx completely occluded inhibition of I _Ca by -CgTx. However, when applied after -CgTx, -GsTx produced an additional inhibition of 27%, indicating that -GsTx also blocked a non-N-type Ca²⁺ channel. BayK8644 (1 M) elicited an increase in I _Ca in the presence of maximally effective concentrations of -GsTx, suggesting that -GsTx does not block L-type channels. Thus, -GsTx displays a selectivity for Ca²⁺ channel subtypes which should prove useful for studying Ca²⁺ channels and Ca²⁺-channel-mediated processes.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.