Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre-and postsynaptic calcium fluxes in rat hippocampal slices

The contribution of T-, L-, N-, P-, and Q-type Ca²⁺ channels to pre-and postsynaptic Ca²⁺ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca²⁺ concentration ([Ca²⁺]₀). [Ca²⁺]₀ was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca²⁺ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca²⁺]₀ decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca²⁺ entry (Ca_pre), while [Ca²⁺]⁰ decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca²⁺ entry (Ca_post). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Ca_post and, much less, also Ca_pre Nimodipine (25 M) reduced Ca_post and, to a minor extent, Ca_pre. -Agatoxin IVA (0.4–1 M) and -conotoxin MVIIC (1 M) also reduced both Ca_pre and Ca_post, but with a stronger action on Ca_pre. -Conotoxin GVIA (3–8 M) reduced Ca_post without effect on Ca_pre. We conclude that during stimulus-induced, high-frequency neuronal activity Ca_post is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Ca_pre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Ca_pre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca²⁺ entry in this situation.     

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.     

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.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

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.     

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.     

Go transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons

High-voltage-activated (HVA) calcium channel currents (I _Ba) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. I _Ba was irreversibly inhibited by 56.9±2.7% by 1 M -conotoxin-GVIA (-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective -aminobutyric acid_B (GABA_B) agonist, (–)-baclofen (50 M), inhibited the HVA I _Ba by 30.7±5.4%. Prior application of -CTx-GVIA completely occluded inhibition of the HVA I _Ba by (–)-baclofen, indicating that in this preparation (–)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G_o antiserum, raised against the C-terminal decapeptide of the G _o subunit, reduced (–)-baclofen inhibition of the HVA I _Ba, whereas replating DRGs in the presence of the anti-G_i antiseram did not. Using anti-G _o antisera (12000) and confocal laser microscopy, G _o localisation was investigated in both unreplated and replated neurons. G _o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G _o immunoreactivity was completely blocked by preincubation with the immunising G_o undecapeptide (1 g · ml^–1) for 1 h at 37° C. A similar treatment also blocked recognition of G _o in brain membranes on immunoblots. These results provide evidence that GABA_B inhibition of N-type calcium channels in acutely replated DRGs occurs via G _o.     

Biphasic short-circuit current response to noradrenaline mediated by Ca2+ and cAMP in cultured rat epididymal epithelium

A study was carried out to investigate the short-circuit current (I _sc) response to noradrenaline (NA) and the signal transduction mechanisms involved in cultured rat cauda epididymal epithelium. In normal Krebs-Henseleit solution, NA (10 mol · l^–1) added basolaterally elicited a biphasic I _sc response consisting of a transient spike followed by a second sustained response. The biphasic response was almost abolished by removing ambient Cl^–. Preloading the tissues witha cell-permeant Ca²⁺ chelator, 1,2-bis(2-aminophenoxy) eth-ane-N,N,N,N,-tetraacetic acid acetoxymethyl ester (BAPTA/AM), or pretreating them with thapsigargin (Tg), a microsomal adenosine triphosphatase inhibitor abolished the initial spike in the I _sc response to NA, but had little effect on the second component. Pretreating the tissues with a non-selective -antagonist, nadolol, reduced the second I _sc response in a dose-dependent fashion but the initial spike was not affected. Microfluorimetric studies showed that NA (100 mol · l^–1) elicited single Ca²⁺ spikes in isolated epididymal cells, which could be abolished by prior treatment with Tg. Biochemical assays showed that NA (10 mol · l^–1) increased intracellular cyclic adenosine monophosphate concentration ([cAMP]_i) and the response was abolished by prior treatment with nadolol (50 mol · l^–1). The results showed that NA elicited a biphasic I _sc response mediated by a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a rise in [cAMP]_i. The Ca²⁺-mediated I _sc response had a faster onset and more transient action than the cAMP counterpart. It is suggested that NA released from noradrenergic nerve endings regulates transepithelial Cl^– secretion in the epididymis thereby providing the specialized millieu vital for sperm storage and maturation.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

A cyclin protein modulates mitosis in the budding yeast Saccharomyces cerevisiae

Summary For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step start. The threshold amount of cell mass (reflected as a critical size) necessary for start is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at start, termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for start, causing the smaller cells, produced in the previous cell cycle, to be delayed at start while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and start, nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

TTX-sensitive Na+ channels and Ca2+ channels of the Land N-type underlie the inward current in acutely dispersed coeliac-mesenteric ganglia neurons of adult rats

Inward membrane currents of sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were characterized using the whole-cell variant of the patch-clamp technique. Current-clamp studies indicated that C-SMG neurons retained electrical properties similar to intact ganglia. Voltage-clamp studies designed to isolate Na⁺ currents revealed that tetrodotoxin (TTX, 1 M) completely inhibited the large transient inward current. Half activation potential (V _h) and slope factor (K) were –26.8 mV and 6.1 mV, respectively. Inactivation parameters for V _h and K were –65 mV and 8.2 mV, respectively. Voltage-clamp studies also revealed a high-voltage-activated sustained inward Ca²⁺ current which was blocked by the removal of external Ca²⁺ or the presence of Cd²⁺ (0.1 mM). The dihydropyridine agonist, (+)202–791 (1 M), caused a small increase (20%) in the amplitude of the Ca²⁺ current at more negative potentials and markedly prolonged the tail currents. -Conotoxin GIVA (, CgTX, 15 M) caused a 66% inhibition of the high-voltage-activated Ca²⁺ current amplitude. Norepinephrine (1 M) caused a 49% reduction in the peak Ca²⁺ current. This study is the first demonstration that dispersed C-SMG neurons from adult rats retain electrical characteristics similar to intact ganglia. A TTX-sensitive Na⁺ current as well as a high voltage-activated sustained Ca²⁺ current underlie the inward current in C-SMG neurons. The macroscopic Ca²⁺ current is composed of a small dihydropyridinesensitive (L-type current) and a large -CgTx-sensitive (N-type current) component. Thus, acutely dispersed CSMG neurons are suitable for examining the biophysical properties and modulation of membrane currents of adult prevertebral sympathetic neurons in normal and diseased states.     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

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.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

β-Adrenergic modulation of transient inward current in guinea-pig cardiac myocytes

Transient inward current (I_ti) indicating Ca²⁺-release from the sarcoplasmic reticulum and L-type Ca²⁺-current(I_Ca) were studied in atrial and ventricular myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. The increase of I_Ca caused by -adrenergic stimulation using isoprenaline (ISO) or related experimental manoeuvres such as superfusion with forskoline (FORSK) was used as a qualitative monitor of an increase of intracellular cAMP. Changes of I_ti were used to manifest changes of sarcoplasmic Ca²⁺-release. In myocytes dialysed with citrate-based (60 mM) pipette filling solution containing 100 M EGTA spontaneous transient inward currents were recorded at a constant holding potential of –50 mV in the majority of myocytes. Superfusion with a solution containing ISO (5·10^–8M) increased the amplitude of spontaneous I_ti and reduced its time-to-peak. The effects of ISO on I_ti developed in parallel to stimulation of I_Ca. In myocytes which did not show spontaneous cyclic Ca²⁺-release in the above condition, this could be evoked de novo by ISO. Spontaneous I_ti was suppressed in the majority of cells by increasing the concentration of EGTA in the dialysing solution to 200 M. Brief (50 ms) activation of I_Ca by voltage steps from –50 to +10 mV usually failed to trigger Ca²⁺-release from the SR. The increase of I_Ca-amplitude upon administration of ISO went ahead with the induction of Ca²⁺-release by brief activation of I_Ca. The effects of ISO could be mimicked by FORSK or intracellular dialysis with 35-cyclic adenosine monophosphate. The effects on I_Ca and SR Ca²⁺-release were dependent on the concentration of the stimulating substance. In a given cell changing superfusion from a low to a high concentration of ISO or FORSK resulted in an increase of the number of Ca²⁺-release events per number of Ca²⁺-currents elicited and a shortening of time-to-peak of I_ti's. The stimulating effects of ISO or FORSK on Ca²⁺-release were only partially due to an increase of the triggering I_Ca. Ca²⁺-currents too small to trigger Ca²⁺-release before -adrenergic stimulation could evoke Ca²⁺-release after augmentation of intracellular cAMP. Whereas the effects of ISO and FORSK on I_Ca were reversible, the stimulatory effects on Ca²⁺-release persisted after washing out the substances. The results give support to the hypothesis that -adrenoceptor-mediated positive inotropic and arrhythmogenic effects are, at least partly, due to a cyclic AMP-dependent regulatory mechanism modulating sarcoplasmic Ca²⁺-release.This work was supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

Inhibition of L-type calcium channels by internal GTP [γS] in mouse pancreatic β cells

Pretreatment of pancreatic cells with pertussis toxin resulted in a 30% increase in peak whole-cell Ca²⁺ currents recorded in the absence of exogenous intracellular guanine nucleotides. Intracellular application of 90 M GTP[S], by liberation from a caged precursor, resulted in 40% reduction of the peak Ca²⁺ current irrespective of whether the current was carried by Ca²⁺ or Ba²⁺. Effects on the delayed outward K⁺ current were small and restricted to a transient Ca²⁺-dependent K⁺ current component. Inhibition by GTP[S] of the Ca²⁺ current was not mimicked by standard GTP and could not be prevented either by pretreatment with pertussis toxin or by inclusion of GDP[S] or cyclic AMP in the intracellular medium. The inhibitory effect of GTP[S] could be counteracted by a prepulse to a large depolarizing voltage. A similar effect of a depolarizing prepulse was observed in control cells with no exogenous guanine nucleotides. These observations indicate that inhibition of cell Ca²⁺ current by G protein activation results from direct interaction with the channel and does not involve second-messenger systems. Our findings also suggest that the cell Ca²⁺ current is subject to resting inhibition by G proteins.     

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1) and tumor necrosis factor alpha (TNF) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1 and TNF were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 and TNF significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 and TNF displayed significant in vivo antitumor activity against the IL1- and TNF-resistant B16F10 metastatic murine melanoma.