Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca²⁺ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of –80 mV, using 10 mM Ba²⁺ as the charge carrier, only high-voltage-activated (HVA) Ca²⁺ channels were found. Ba²⁺ currents (I _Ba) snowed variable sensitivity to dihydropyridine (DHP) Ca²⁺ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca²⁺ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I _Ba was accompanied by a 20-mV shift in the activation curves for Ca²⁺ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I _Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I _Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I _Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca²⁺ channels. The sequential addition of supramaximal concentrations of furnidipine (10 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I _Ba, which were additive. Our data suggest that the whole cell I _Ba in rat chromaffin cells exhibits at least four components. About 50% of I _Ba is carried by L-type Ca²⁺ channels, 30% by N-type Ca²⁺channels and 15% by P-type Ca²⁺ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca²⁺channels account for 45% of the current, N-type carry 35% and L-type Ca²⁺ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.     

Dihydropyridines,phenylalkylamines and benzothiazepines block N-, P/Q- and R-type calcium currents

We compared the effects of representative members of three major classes of cardiac L-type channel antagonists, i.e. dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BTZs) on high-voltage-activated (HVA) Ca²⁺ channel currents recorded from a holding potential of –100 mV in rat ventricular cells, mouse sensory neurons and rat motoneurons. Nimodipine (DHP), verapamil (PAA) and diltiazem (BTZ) block the cardiac L-type Ca²⁺ channel current (EC₅₀: 1 M, 4 M and 40 M, respectively). At these concentrations, the drugs could also inhibit HVA Ca²⁺ channel currents in both sensory and motor neurons. Large blocking effects (> 50%) could be observed at 2–10 times these concentrations. The -conotoxin-GVIA-sensitive (-CTx-GVIA, N-type), -agatoxin-IVA-sensitive (-Aga-IVA, P- and Q-types) and non-L-type -CTx-GVIA-, -Aga-IVA-insensitive (R-types) currents accounted for more than 90% of the global current. Furthermore, our data showed that CTx-GVIA and -Aga-IVA spare L-type currents and have only additive blocking effects on neuronal HVA currents. We conclude that DHPs, PAAs and BTZs have substantial inhibitory effects on neuronal non-L-type Ca²⁺ channels. Inhibitions occur at concentrations that are not maximally active on cardiac L-type Ca²⁺ channels.     

Voltage-dependent noradrenergic modulation of ω-conotoxin-sensitive Ca2+ channels in human neuroblastoma IMR32 cells

High-threshold (HVA) Ca²⁺ channels of human neuroblastoma IMR32 cells were effectively inhibited by noradrenaline. At potentials between –20 mV and +10 mV, micromolar concentrations of noradrenaline induced a 50%–70% depression of HVA Ba²⁺ currents and a prolongation of their activation kinetics. Both effects were relieved at more positive voltages or by applying strong conditioning pre-pulses (facilitation). Facilitation restored the rapid activation of HVA channels and recruited about 80% of the noradrenaline-inhibited channels at rest. Re-inhibition of Ca²⁺ channels after facilitation was slow ( _r 36–45 ms) and voltage-independent between –30 mV and –90 mV. The inhibitory action of noradrenaline was dose-dependent (IC₅₀=84 nM), mediated by ₂-drenergic receptors and selective for -conotoxin-sensitive Ca²⁺ channels, which represent the majority of HVA channels expressed by IMR32 cells. The action of noradrenaline was mimicked by intracellular applications of GTP[S] and prevented by GDP[S] or by pre-incubation with pertussis toxin. The time course of noradrenaline inhibition measured during fast application (onset) and wash-out (offset) of the drug were independent of saturating agonist concentrations (10–50 M) and developed with mean time constants of 0.56 s ( _on) and 3.6 s ( _off) respectively. The data could be simulated by a kinetic model in which a G protein is assumed to modify directly the voltage-dependent gating of Ca²⁺ channels. Noradrenaline-modified channels are mostly inhibited at rest and can be recruited in a steep voltage-dependent manner with increasing voltages.     

Inhibition of ω-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones

The effects of changing the intracellular concentrations of either free Mg²⁺ ions ([Mg²⁺]_i) or Mg²⁺-bound adenosine triphosphate ([Mg · ATP]_i) on Ca²⁺ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg²⁺]_i from 0.06 mM to 1.0 mM inhibited Ca²⁺ channel currents by approximately 50%. The action of -conotoxin GVIA (-CgTX), a selective inhibitor of N-type Ca²⁺ channels was also investigated. With increasing [Mg²⁺]_i, the proportion of current irreversibly blocked by -CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg²⁺]_i values of 0.5 mM or greater. Block of the -CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg²⁺]_i to 0.5 mM. Raising [Mg²⁺]_i had no effect on the rate of decay of Ca²⁺ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg · ATP]_i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to -CgTX. [Mg²⁺]_i was also effective in inhibiting the Ca²⁺ channel current following potentiation by increasing [Mg · ATP]_i. These data suggest that -CgTX-sensitive current in these cells is selectively inhibited by internal Mg²⁺ whereas both -CgTX-sensitive and -resistant components of current are potentiated by internal Mg · ATP. The mechanism by which Mg²⁺ inhibits N-type channels is unclear, but may involve an open channel block.     

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.     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

Zum Mechanismus der ökonomischen Halteleistung eines glatten Muskels (Byssus retractor anterior,Mytilus edulis)

Summary Freeze-dried fibre-bundles (GF) of the anterior byssus retractor muscle (ABRM) ofMytilus develop in Ca²⁺-containing ATP-salt-solution almost the same isometric tension as the surviving muscle and maintain the same tension remnant when Ca²⁺ is deprived. The ratio ATPase/tension of Ca²⁺-activated GF (0.15 mcal/sec × kg tension × cm fibre at 20°C, determined from ATPase measurements) corresponds to the heat of maintenance of living muscle in tetanus (0.07 mcal/see × kg × cm), while it is nearly zero when GF are in the catch state in Ca²⁺-free solution.Contrary to isometric tension and tension remnant, the energy expenditure and speed of tension development depend on temperature (Q ₁₀3); moreover the ratio ATPase/tension of Ca²⁺-activated GF decreases to 70% as the extent of the caught state increases in the course of three successive periods of contraction. But it increases again to the former level when the catch is abolished.These findings are discussed in relation to the Paramyosin-theory of catch. With regard to the typical ultrastructure of caught muscle fibres it is suggested that their increased economy is based on interaction of the thick paramyosinfilaments.

     

Expression of calcium channel subunits in the normal and diseased human myocardium

We investigated the expression of ₁ and subunits of the L-type Ca²⁺ channel on the protein level in cardiac preparations from normal human heart ventricles and from the hypertrophied septum of patients with hypertrophic obstructive cardiomyopathy (HOCM). 1,4-Dihydropyridine (DHP) binding and immunorecognition by polyclonal antibodies directed against the C-terminal amino acid sequences of the ₂ and ₃ subunits were used for detection and quantification of ₁, ₂, and ₃ subunits. B_max of high-affinity DHP binding was 35±2 fmol/mg protein in HOCM and 20±2 fmol/mg protein in normal human hearts (P<0.05). In rabbit hearts the anti-₂ subunit antibody immunoprecipitated 80% of the total amount of DHP-labeled Ca²⁺ channels present in the assay. Under identical experimental conditions 25% of labeled Ca²⁺ channels were recovered in the immunoprecipitates of both normal and HOCM ventricles. A similar partial immunoprecipitation was observed in pig hearts. Immunoblot analysis demonstrated that the ₂ subunit was associated with the DHP receptor/Ca²⁺ channel in cardiac muscle of rabbit, pig, and human heart. In neither of these purified cardiac Ca²⁺ channels was the ₃ subunit isoform detected. Our results suggest that both ₁ and ₂ subunit expression is upregulated in HOCM in a coordinate manner.Abbreviations B _max Maximal number of binding sites - DHP 1,4-Dihydropyridine - HOCM Hypertrophic obstructive cardiomyopathy - NH Normal human heart     

A single non-L-, non-N-type Ca2+ channel in rat insulin-secreting RINm5F cells

Single high-voltage-activated (HVA) Ca²⁺ channel activity was recorded in rat insulinoma RINm5F cells using cell-attached and outside-out configurations. Single-channel recordings revealed three distinct Ca²⁺ channel subtypes: one sensitive to dihydropyridines (DHPs)-(L-type), another sensitive to -conotoxin (CTx)-GVIA (N-type) and a third type insensitive to DHPs and -CTx-GVIA (non-L-, non-N-type). The L-type channel was recorded in most patches between –30 and +30 mV The channel had pharmacological and biophysical features similar to the L-type channels described in other insulin-secreting cells (mean conductance 21 pS in control conditions and 24 pS in the presence of 5 M Bay K 8644). The non-L-, non-N-type channel was recorded in cells chronically treated with -CTx-GVIA in the presence of nifedipine to avoid the contribution of N- and L-type channels. Channel activity was hardly detectable below –10 mV and was recruited by negative holding potentials (< –90 mV). The channel open probability increased steeply from –10 to +40 mV Different unitary current sublevels could be detected and the current voltage relationship was calculated from the higher amplitude level with a slope conductance of 21 pS. Channel activity lasted throughout depolarizations of 300–800 ms with little sign of inactivation. Above 0 mV the channel showed a persistent flickering kinetics with brief openings (₀ 0.6 ms) and long bursts (_burst 60 ms) interrupted by short interburst intervals. The third HVA Ca²⁺ channel subtype, the N-type, had biophysical properties similar to the non-L-, non-N-type and was best identified in outside-out patches by its sensitivity to -CTx-GVIA. The channel was detectable only above –10 mV from a –90 mV holding potential, exhibited a fast flickering behaviour, persisted during prolonged depolarizations and had a slope conductance of about 19 pS. The present data provide direct evidence for a slowly inactivating non-L-, non-N-type channel in insulin-secreting RINm5F cells that activates at more positive voltages than the L-type channel and indicate the possibility of identifying unequivocally single HVA Ca²⁺ channels in cell-attached and excised membrane patches under controlled pharmacological conditions.     

Block of non-L-, non-N-type Ca2+ channels in rat insulinoma RINm5F cells by ω-agatoxin IVA and ω-conotoxin MVIIC

The high-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma RINm5F cells insensitive to dihydropyridines (DHP) and -conotoxin GVIA (-CTx-GVIA) have been studied for their sensitivity to -agatoxin-IVA (-Aga-IVA) and -CTx-MVIIC. Blockade of HVA currents by -Aga-IVA was partial (mean 24%), reversible and saturated around 350 nM (half block 60 nM). Blockade by -CTx-MVIIC was more potent (mean 45%), partly irreversible and saturated above 3 M. The effects of both toxins were additive with that of nifedipine (5 M) and were more pronounced at positive potentials. -Aga-IVA action was additive with that of -CTx-GVIA (3 M) but was largely prevented by cell pre-treatment with -CTx-MVIIC (3 M). In contrast, -CTx-MVIIC block was attenuated by -CTx-GVIA treatment ( 15%), suggesting that -CTx-MVIIC blocks the N-type ( 15%) and the non-L-, non-N-type channel sensitive to -Aga-IVA ( 30%). Consistent with this, cells deprived of most non-L-type channels by pre-incubation with -CTx-GVIA and -CTx-MVIIC exhibited predominant L-type currents that activated at more negative potentials than in normal cells (-30 mV in 5 mM Ba²⁺) and were effectively depressed by nifedipine (maximal block of 95% from -30mV to +40 mV). Our results suggest that, besides L- and N-type channels, insulin-secreting RINm5F cells possess also a non-L-, non-N-type channel that contributes significantly to the total current ( 30%). Although the pharmacology of this channel is similar to Q-type and ₁ class A channels, its range of activation (>-20 mV) and its slow inactivation time course resemble more that of N- and P-type channels. The channel is therefore referred to as Q-like.     

Ca currents in human neuroblastoma IMR32 cells: kinetics,permeability and pharmacology

We have investigated the kinetics, permeability and pharmacological properties of Ca channels in in vitro differentiated IMR32 human neuroblastoma cells. The lowthreshold (LVA, T) Ca current activated positive to –50 mV and inactivated fully within 100 ms in a voltage-dependent manner. This current persisted in the presence of 3.2 M -conotoxin (-CgTx) or 40 M Cd and showed a weaker sensitivity to Ni and amiloride than in other neurons. The high-threshold Ca currents (HVA,L and N) turned on positive to –30 mV, and inactivated slowly and incompletely during pulses of 200 ms duration. The amplitude of the HVA currents and the number of ¹²⁵I--CgTx binding sites increased markedly during cell differentiation. In agreement with recent reports, 6.4 M -CgTx blocked only about 85% of the Ba currents through HVA channels in 50% of the cells. Residual -CgTx-resistant currents proved to be more sensitive to dihydropyridines (DHP) than total HVA currents. Bay K 8644 (1 M) had a clear agonistic action on -CgTx-resistant currents and was preferred to other Ca antagonists for identifying HVA DHP-sensitive channels. Compared to the -CgTx-sensitive, the DHP-sensitive currents turned on at slightly more negative potentials and showed a weaker sensitivity to voltage. The two HVA currents were otherwise hardly distinguishable in terms of activation/inactivation kinetics, Ca/Ba permeability and sensitivity to holding potentials. This suggests that currently used criteria for identifying multiple types of neuronal Ca channels (T,L,N) may be widely misleading if not supported by pharmacological assays.     

A Gi1–2-protein is required for α2A-adrenoceptor-induced stimulation of voltage-dependent Ca2+ channels in rat portal vein myocytes

In rat portal vein myocytes, _2A-adrenoceptors activate voltage-dependent Ca²⁺ channels via a transduction pathway requiring protein kinase C activation mediated by a pertussis-toxin-sensitive G-protein. As revealed by the use of antibodies directed against the different -subunits expressed in portal vein myocytes, we show that the clonidine-induced stimulation of voltage-dependent Ca²⁺ channels is mainly mediated by a G_i1–2-protein.     

Ca2+ not cyclic AMP mediates the fluid secretory response to isoproterenol in the rat mandibular salivary gland: Whole-cell patch-clamp studies

We have performed whole-cell patch-clamp studies on dispersed seccretory cells of the rat mandibular gland to determine how -adrenergic stimulation causes fluid secretion. When the pipette contained a high K⁺ solution, the resting membrane potential averaged –33 mV±1.1 (SEM,n=34) and the clamped cell showed strong outward rectification. We monitored K⁺ and Cl^– currents for periods of 15 min by recording the currents needed to clamp the cell potential at 0 and –80 mV, respectively. Isoproterenol (1–2 mol/l) caused increases in the clamp current at 0 mV (the K⁺ current) and at –80 mV (the Cl^– current) in about 80% of cases, although the responses were variable in size and time-course; the responses were indistinguishable from those induced by acetylcholine or the Ca²⁺ ionophore, A23187. The -adrenergic antagonist, phentolamine (1–2 mol/l), had no effect on the response, but the -adrenergic antagonist, propranolol (10 mol/l), blocked it completely. The isoproterenol response could not be mimicked by application to either surface of the cell membrane, of cyclic AMP (100 mol/l), forskolin (1 or 20 mol/l) or cholera toxin (2.5 g/ml). However, increasing the Ca²⁺-chelating capacity of the pipette solution by raising its EGTA concentration from the customary 0.5 to 20 mmol/l, blocked the response to isoproterenol, suggesting that -adrenergic agonists activate Cl^– and K⁺ channels by raising cytosolic Ca²⁺. Since neomycin, which blocks phospholipase C, blocked the action of isoproterenol without impairing the cell responsiveness to A23187, it appears that isoproterenol, like muscarinic agonists, increased cytosolic Ca²⁺ via the phosphatidylinositol cycle.This project was supported by the National Health and Medical Research Council of Australia     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

The sensitivity of pineal glandβ-receptors appears to be dependent upon calcium ions

Summary Rat pineals were usedin vitro to demonstrate that calcium antagonists induce a hyposensitive response to stimulation of serotonin N-acetyl transferase (NAT) by the -agonist isoproterenol or by dibutyryl cyclic AMP. Mn²⁺, Co²⁺ and La³⁺ at 10^–3M all significantly reduce the effects of isoproterenol stimulation. In addition, the two enantiomers of D600 also inhibit NAT induction, the D-form slightly more effectively than the L-form. Interestingly, La³⁺ is also able to inhibit NAT after DBcAMP, indicating that Ca²⁺ can also control enzyme induction at an intracellular site beyond the -receptor.     

Ion-induced release of LHRH,TRH and α-MSH from hypothalamic synaptosomes and its inhibition by vinblastine

Homogenates of rat hypothalamic tissue were fractionated by means of discontinuous sucrose density gradient centrifugation. Immunoreactive luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and -melanocyte stimulating hormone (-MSH) were found to be concentrated in the synaptosome-enriched fraction. This fraction was suspended in 0.32 M sucrose and the release of the three peptides was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration, and the concentration of each peptide in the ultrafiltrate was determined by radioimmunoassay. When the synaptosomal fraction was incubated at 30° C in 0.32 M sucrose containing either 60 mM K⁺-2 mM Ca²⁺ or 140 mM Na⁺ alone a release of LHRH, TRH, and -MSH occurred. Of the total content 30–50% of LHRH but only about 10% of TRH and -MSH was releasable. When the synaptosome preparation was preincubated for 30 min at 30°C with 10^–4 M vinblastine. K⁺- as well as Na⁺-induced release of LHRH, THR, and -MSH was inhibited, and the stimulatory effect of each cation was almost totally blocked by preincubation with 5×10^–4 and 10^–3 M vinblastine. The inhibitory action of vinblastine (5×10^–4 M) did not affect the oxidation of glucose to CO₂ by the synaptosomes. The results of the present investigation demonstrate that synaptosome-enriched fractions of hypothalamic origin are more stable with respect to LHRH, TRH, and -MSH release during incubation in isotonic sucrose than they are in ionic solutions, and that the peptides are released by a vinblastine-sensitive mechanism.Supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AMO 1237), the National Institute of Child Health and Human Development (HDO8672), and the National Institutes of Health contract (5-P17-HL1487-06)Supported by Grant No. 512-6951 from the Danish Medical Research Council     

Evidence for a multigenic system controlling methyl-β-carboline-3-carboxylate (β-CCM)-induced seizures

-CCM is a -carboline known to have properties opposite to those of benzodiazepines. Our approach was to analyze, in mice, the genetic mechanisms involved in -CCM-induced myoclonic seizures using recombinant congenic strains and F₁ hybrids issued from these strains. Our aim was to define the extent of the multigenic character of -CCM-induced myoclonic seizures, while also evaluating the distribution of the strength of the genes implicated in this trait. The results show that the control of reactivity to -CCM is multigenic with notable epistatic involvement.     

Interferon β1a Treatment Modulates TH1 Expression in γδ + T Cells from Relapsing-Remitting Multiple Sclerosis Patients

A paradigm exists that multiple sclerosis is causally related to dysregulation of T_H1 inflammatory cytokines and T_H2 antiinflammatory cytokines. The cytokine source(s) that initiate the imbalances are unknown. In this study, , CD4, and CD8 T cell receptor-positive (TCR+) cells were isolated from the blood of 26 definitive relapsing-remitting multiple sclerosis patients prior to interferon -1a (IFN1a) therapy and following 8–10 weeks of this therapy. The bioactivities of interferon (IFN), interleukin 10 (IL10), and interleukin 12 (IL12) were determined. The concentrations of IFN, IL10, and IL12 from each cell type did not change significantly with IFN1a treatment. The IL10 secreted by TCR+ cells strongly correlated with the IL12 secreted by the same TCR+ cells, supporting the paradigm. Furthermore, IFN1a therapy decreased the TCR+ cell secretion of T_H1 cytokines after 8–10 weeks of therapy.     

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.     

Modulation of Ca-channel current by an adenosine analog mediated by a GTP-binding protein in chick sensory neurons

Inhibitory modulation of the high-voltage-activated (HVA) Ca-channel current by 2-chloroadenosine (2CA) was studied in chick sensory neurons using the whole-cell clamp method. 2CA reduced the CTX-sensitive HVA-current (Aosaki and Kasai 1989) in a dose-dependent manner with aK _d of 0.8 M. The inhibition by 2CA was also voltage-dependent, being maximal at hyperpolarized potentials, and completely removed at potentials more positive than 30 mV. This voltage-dependence of 2CA action was also evident as a progressive increase in Ca-channel current magnitude during a depolarization which could be described by a single exponential function and which became faster at larger depolarizations. The concentration of 2CA affected the steady-state reduction in Ca-channel current, but did not alter the time-course of current increase during depolarization. The voltage-dependent effect of 2CA was mimicked by intracellular application of GTP-S, but not by phorbol ester, arachidonic acid or nitroprusside. These results are consistent with model in which 2CA activates a G-protein, which then unmasks an additional activation gate on the Ca-channel.