Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4,5-dimethylfluorescein to investigate intracellular pH (pH_i) regulation in A10 vascular smooth muscle cells: (1) The steady state pH_i in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO₃/5% CO₂). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pH_i-Recovery after an acute intracellular acid load (by means of NH₄Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pH_i recovery increased with increasing sodium concentrations with an apparentK _m for external sodium of about 30 mM and aV _max of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pH_i recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pH_i recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pH_i backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10^–4 M DIDS, suggesting that the effects were mediated by a Cl/HCO₃ exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO₃ exchanger in A10 vascular smooth muscle cells.Abbreviations used CDMF 5(and6)-carboxy-4,5-dimethylfluorescein - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NMDG N-methyl-d-glucamine; pH_i, intracellular pH - pH_o extracellular pH - Mops 3-[N-Morpholino]propanesulfonic acid - Hepes 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - Tris Tris(hydroxymethyl)-aminomethane - EDTA ethylenediamine-tetraacetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)N,N-tetraacetic acid     

A structural model for the genome of echovirus 22

Summary We have proposed previously that the structural model for the echovirus 22 genome is a single-stranded RNA molecule that has folded back upon itself to form a stable hairpin at the 5-terminus. The vRNA of echovirus 22 has been characterized further by digestion with selective ribonucleases, electrophoresis in composite gels, hydrodynamic studies in density gradients of Cs₂SO₄ and sucrose, thermal denaturation and 3-terminal ribonucleotide analysis. Based on these observations, the genome of echovirus 22 is a single-stranded RNA molecule having a region of secondary structure located at the 5-terminus that may be characterized as a snapback hairpin with hydrogen-bonded base-pairing. In addition, a VPg-like protein is attached (presumably to the 5-end of the RNA) and the 3-terminus contains a polyadenylic acid tract [poly(A)].     

Intracellular protons inhibit inward rectifier K+ channel of guinea-pig ventricular cell membrane

The effect of intracellular protons (H_i ⁺) on the inward rectifier K⁺ channel of the guinea-pig ventricular cell membrane was examined, using the patch-clamp technique. The inward single-channel current was recorded in inside-out and outside-out patch configurations, while the pH of the solution perfusing the intra and extracellular side, respectively, was varied. Low intracellular pH (pH_i), but not low extracellular pH, inhibited the channel. Low pH_i reduced the unit amplitude, which was about 20% smaller at pH_i 6.0 than that at pH_i 7.4 at every voltage tested. The slope conductance decreased from 41.7 pS at pH_i 7.4 to 35.1 pS at pH_i 6.0. Low pH_i also reduced the channel activity without apparent voltage dependence. The concentration/response curve indicated the half-maximum inhibition at pH_i 6.11 and a Hill coefficient of 2.52. Lowering the pH_i from 7.4 to 6.0 did not affect the distributions of the open times and the closed times below 50 ms, while the time constant of the histogram constructed from closings longer than 50 ms was approximately doubled. These results indicate that the inward rectifier K⁺ channel in ventricular myocytes is inhibited by H⁺ from the intracellular side. This might contribute to the depolarization of the resting membrane potential induced by intracellular acidosis during myocardial ischaemia.     

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

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

Examination of a purified A′ influenza virus preparation by potentiometry

Summary The potentiometric titration of a purified influenza A virus preparation revealed 100.7×10^–4 M base and 68.8 × 10^–4 M acid-binding capacity per g. of virus protein N. The titration curve was characterized by the following fourpK values:pK ₁ = 3.37;pK ₂ = 4.50;pK ₃ = 6.37, andpK ₄ = 9.75. The isoionic point was at pH 5.43.Attempt was made to identify the dissociating groups and it was found that the carboxylic groups (pK ₁ andpK ₂) may either be glutamyl or aspartyl groups, while the cationic groups are probably the imidazolium of histidine (pK ₃) and the -amino residues of lysine (pK ₄).Inaotivation of the hemagglutinating activity of the virus preparation by mild treatment with formaldehyde at pH 8.0 resulted in a simultaneous disappearence of the -amino groups of lysine (pK ₄). The same treatment at pH 9.0 resulted in the loss of all the cationic groups previously demonstrable.The possible role of the stable positive charges on the surface of the virus at physiological pH is discussed from the point of view of the physico-chemistry of the hemagglutination.     

An investigation of intrinsic buffering power in rat vascular smooth muscle cells

Intrinsic buffering power ( _i) has been measured in vascular strips and single cells from rat mesenteric artery. Intracellular pH (pH_i) regulation was inhibited to prevent overestimation of _i due to acid extrusion or entry via regulatory processes. At resting values of pH_i (7.0–7.2), a mean value of 41±4 mM/pH unit for _i was found. _i increased approximately fivefold from 30 to 150 mM/pH unit over the pH_i range 7.5–6.5. The mean data relating _i to pH_i could be described by relating _i to buffer concentrations and pK _a. This gave a value of 310 mM for buffer concentration and a pK _a of 6.0. As changes in pH_i are known to have marked effects on vascular tone then the increase in _i as pH_i falls may be considered as a means of attenuating pH_i decreases, before pH regulation restores pH_i to resting levels.     

Expression of parathyroid hormone receptors in MDCK and LLC-PK1 cells

Parathyroid hormone (PTH) inhibits renal proximal tubular phosphate (P_i) and bicarbonate reabsorption by regulating the activity of apical Na/P_i cotransport and Na/H exchange. Two renal epithelial cell lines [proximal tubular, LLC-PK₁; distal tubular, Madin-Darby canine kidney, (MDCK) cells] were stably transfected with complementary deoxyribonucleic acids (cDNAs) encoding a cloned PTH receptor in order to examine the polarity of transfected receptor function and whether or not intrinsic P_i transport is regulated by the transfected PTH receptor. The receptors are functionally coupled to the stimulation of adenosine 35 cyclic monophosphate (cAMP) production at both cell surfaces in LLC-PK₁ cells, whereas this response is primarily limited to the basolateral surface in MDCK cells. Immunocytochemistry suggests an apical and basolateral localization of the transfected PTH receptor in LLC-PK₁ cells and only a basolateral localization in MDCK cells. PTH activation of the transfected receptors is not coupled to the regulation of intrinsic P_i transport in either LLC-PK₁ or MDCK cells.     

Characterization of the hog cholera virus 5′ terminus

Hog cholera virus (HoCV) 5 terminus of the ALD and GPE(–) strains were analyzed by using rapid amplification of cDNA end method (5RACE). An additional nine nucleotides were found at the 5 termini of genomic RNA in the ALD and GPE(–) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5 terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5 terminus sequence has a significant role in the HoCV virus genome.     

Methoden zur Bestimmung des Lichtweges bei der Photometrie trüber Lösungen oder Gewebe mit durchfallendem oder reflektiertem Licht

Summary Reflexion spectra of living organs are not linearly transformed as compared to multiplicatively mixed spectra of transparent solutions having both uniform concentration and composition of the components. From this non-linear transformation inhomogeneous light paths can be assumed. The present paper described two methods which permit to calculate the probability function for the path covered in the probe.The first method uses an approximation of the light path distribution by discontinuous functions, whereas with the second method the light path distribution is developed in a sequence with normalized polynomials. The addition of a known dye to the tissue permits to calculate the dependence of the light path distribution on the wavelength.As an example, the probability function is calculated for the way covered in the guinea-pig-brain.

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Erklärung der benutzten Symbole A () additiv gemischtes Spektrum in Abhängigkeit von der Wellenlänge - B (N+1)-zeilige und einspaltige Matrix aus 10^{–H(i h)} - B ^T transformierte (gespiegelte) Matrix vonB - c _i Konzentrationswerte - h Parameter - H (M) Transformation zwischen multiplikativer und additiver Farbstoffmischung - H (M), H (M) erste bzw. zweite Ableitung vonH (M) - h ₀ Cuvettenhöhe - i Parameter aus natürlichen Zahlen oder Index - K Zahl der Komponenten eines Mehrkomponentenspektrums - L () Intensität des durch eine Cuvette der Dickez ₀ gefallenen Lichtes - L ₀ Intensität des einfallenden Lichtes - l (M) reflektierte Lichtintensität in Abhängigkeit vonM - l ₀ an einem nicht absorbierenden Vergleichsobjekt reflektierte Lichtintensität - lgx=log₁₀ x Zehnerlogarithmus - M () multiplikativ gemischtes Spektrum, bezogen auf einen Lichtwegz ₀ - M ₂,M ₁ größter bzw. kleinster Wert, für denH (M) bekannt ist - N nachN+1 Gliedern wird die Reihenentwicklung von (x) abgebrochen - n Parameter - P _v (x) Legendre Polynome - q _v Koeffizienten der Reihenentwicklung von (x) nach den PolynomenU _v (x) - q (N+1)-zeilige und einspaltige Matrix aus den Wertenq _v - q ^T transformierte (gespiegelte) Matrix vonq - r _v durch definiert - U _v (x) im Intervall (0,1) orthonormierte Polynome - U (N+1)-zeilige und einspaltige Matrix aus den PolynomenU _v (x) - V (N+1)×(N+1) Matrix, definiert durchU=V x - V ^T transformierte (gespiegelte) Matrix vonV - W (z) Wahrscheinlichkeit, daß in der Probe ein Lichtweg <z zurückgelegt wurde - (x) Verteilungsfunktion in Abhängigkeit vonx - x alsx=10^– hz/z _o definiert - x (N+1)-zeilige und einspaltige Matrix aus den Wertenx ⁱ - z Lichtweg - mittlerer Lichtweg - z Abweichungen vom mittleren Lichtweg - mittlere quadratische Abweichung - z (W) Lichtweg in Abhängigkeit von der WahrscheinlichkeitW, Umkehrfunktion vonW (z) - z _v s. Formel (15) - z ₀ Bezugswert für den Lichtweg - Anteil des bei absorptionsfreiem Vergleichsobjekt in den Empfänger gelangtes Licht - (y) Heavisidesche Sprungfunktion - _i () molare Extinktionskoeffizienten in Abhängigkeit von der Wellenlänge - Wellenlänge - , Index aus natürlichen Zahlen - (z) Wahrscheinlichkeitsdichtefunktion für den zurückgelegten Lichtwegz     

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

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

Role of mast cells in calcium ionophore (A23187)-induced peritoneal inflammation in mice

Several lines of evidence document a critical role for mast cells in immune complex-mediated inflammatory models. However, their role in nonimmune models of acute inflammation is largely unknown. In the present investigation, the role of mast cells was examined in calcium ionophore (A23187)-induced mouse peritoneal inflammation. Intraperitoneal injection of A23187 (20)g/mouse) elicited marked and transient increases in immunoreactive levels of 6-ketoprostaglandin-F₂, leukotrienes B₄, C₄, D₄, E₄, and F₄. There were no discernible differences in levels of these mediators in male Swiss Webster mice, mast cell-deficient mice (WBB6F1-W/W), and age-matched controls (WBB6F1-+/+), suggesting a minimal role of mast cells in eicosanoid biosynthesis in this model. However W/W mice showed smaller increases in levels of myeloperoxidase, a marker for neutrophils, compared to +/+ mice. Both W/W and +/+ mice have lower constitutive levels of peritonealN-acetyl--d-glucosaminidase (NAG), a marker for mononuclear cells. Similar to the changes seen in myeloperoxidase, W/W mice exhibited a blunted NAG response compared to +/+ mice. These results suggest that mast cell products other than eicosanoids may contribute to the changes in cellular trafficking in response to intraperitoneal A23187. These results also suggest that mast cells are required for full expression of inflammatory responses.     

Intracellular pH in renal tubules in situ: single-cell measurements by confocal laserscan microscopy

Confocal laserscan microscopy with a dual-excitation device was used to record intracellular pH (pH_i) regulation in rat proximal convoluted tubules microperfused in vivo. Cells were loaded with the pH-sensitive, fluorescent dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Single cells could be distinguished within the tubules and separate measurements were possible. Application of an NH₄Cl pulse by peritubular perfusion caused an immediate increase in intracellular pH. Intraluminal injection of NH₄Cl led to a slower increase in intracellular pH. In both cases, cessation of perfusion led to an immediate acidification. Peritubular perfusion with 300 M 4,4-diisothiocanatodihydrostilbene-2, 2-disulphonic acid (H₂DIDS) caused an intracellular alkalinisation. Microperfusion, pH-sensitive dyes and confocal laserscan microscopy provide a new non-invasive method to measure intracellular pH effectively in individual cells of near-surface structures of the intact kidney.     

Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells

The mechanism of regulation of intracellular pH (pH_i) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2,7-bis(2-carboxyethyl)-5(and –6)-carboxyfluorescein. The pH_i in the TRIS/HEPES-buffered standard solution was 7.29±0.01. Addition of 1 mol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pH_i. These agents decreased pH_i in the absence of external Na⁺ or in the presence of amiloride. The rate of pH_i recovery from an acid load after NH ₄ ⁺ prepulse was a linear function of pH_i and increased as pH_i became more acidic. Addition of ACh shifted the relationship towards a more alkaline pH_i range. The increase in pH_i induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 mol/l). Addition of 0.1–1 mol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pH_i within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pH_i. In Ca²⁺-free solution with 50 mol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pH_i and cytosolic Ca²⁺ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 mol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na⁺/H⁺ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na⁺/H⁺ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na⁺/H⁺ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca²⁺ concentration. The implication of the cytosolic acidification and cell volume change in pH_i regulation is discussed.     

Translation enhancer in the 3′-untranslated region of rotavirus gene 6 mRNA promotes expression of the major capsid protein VP6

Summary. The eleven rotavirus mRNAs contain 5-cap structures and most end with the 3-consensus sequence 5-UGACC-3. The UGACC functions as a common translation enhancer (3-TE-con) that upregulates viral protein expression through a process mediated by the nonstructural protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5-and 3-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3-truncations showed that a highly active enhancer was present near the 5-end of the 139-nucleotide 3-UTR of the gene 6 mRNA (3-TEg6). The 3-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3-TEg6 differs significantly from the 3-TE-con in that the gene 6-specific enhancer does not require viral protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3-TEg6) and the other is common to nearly all rotavirus genes (3-TE-con). The activity of the 3-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.     

Studien über den Mechanismus der Immunhämolyse: Der Bindungsmodus der vierten Komplementkomponente

Zusammenfassung Die Bindung der vierten Komplementkomponente (C₄) an sensibilisierte Hammelblutzellen (EA) ist von der vorhergehenden Bindung der ersten Komplementkomponente abhängig. Das Präparat EAC₁ kann, wenn ihm C₄ in geeigneter Form angeboten wird, in den Komplex EAC_1,4 überführt werden. Hierbei verschwindet der titrierbare Gehalt der flüssigen Phase an C₄; gleichzeitig acquirieren die Zellen die Fähigkeit, mit R₄ zu lysieren. Der zeitliche Ablauf dieser Veränderung wird untersucht. Die Eeaktion zwischen EAC₁ und C₄ verläuft äußerst schnell, und ihre Geschwindigkeit wird durch die Reaktionstemperatur nicht beeinflußt. Während die Bindung von C₁ an EA nur in Gegenwart von Ca⁺⁺ erfolgt, läuft die Bindung von C₄ an EAC₄ auch in Abwesenheit von zweiwertigen Metallionen ab. Die vonLevine u.Mayer als ein Ganzes betrachtete Ca⁺⁺-abhängige Überführung von EA in EAC_1,4 besteht mithin aus zwei aufeinanderfolgenden Teilreaktionen, von denen die erste (C₁-Bindung) calciumabhängig und die zweite (C₄-Bindung) von zweiwertigen Ionen unabhängig ist.Mit Unterstützung der Deutschen Forschungsgemeinschaft sowie der Gesellschaft der Freunde und Förderer der Medizinischen Akademie Düsseldorf.     

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.     

Sensitivity to dihydropyridines, ω-conotoxin and noradrenaline reveals multiple high-voltage-activated Ca2+ channels in rat insulinoma and human pancreatic β-cells

High-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma (RINm5F) and human pancreatic -cells were tested for their sensitivity to dihydropyridines (DHPs), -conotoxin (-CgTx) and noradrenaline. In RINm5F cells, block of HVA currents by nimodipine, nitrendipine and nifedipine was voltage- and dose-dependent (apparent K _D<37 nM) and largely incomplete even at saturating doses of DHPs (mean 53%, at 10 M and 0 mV). Analysis of slow tail currents in Bay K 8644-treated cells indicated the existence of Bay K 8644-insensitive channels that turned on at slightly more positive voltages and deactivated more quickly than Bay K 8644-modified channels. DHP Ca²⁺ agonists and antagonists in human -cells had similar features to RINm5F cells except that DHP block was more pronounced (76%, at 10 M and 0 mV) and Bay K 8644 action was more effective, suggesting a higher density of L-type Ca²⁺ channels in these cells. In RINm5F cells, but not in human -cells, DHP-resistant currents were sensitive to -CgTx. The toxin depressed 10–20% of the DHP-resistant currents sparing a residual current (25–35%) with similar voltage-dependent characteristics and Ca²⁺/Ba²⁺ permeability. Noradrenaline (10 M) exhibited different actions on the various HVA current components: (1) it prolonged the activation kinetics of -CgTx-sensitive currents, (2) it depressed by about 20% the size of DHP-sensitive currents, and (3) it had little or no effects on the residual DHP- and -CgTx-resistant current although intracellularly applied guanosine 5-O-(3-thiotriphosphate) (GTP--S) prolonged its activation time course. The first action was clearly voltage-dependent and most evident in RINm5F cells that displayed neuronal-like processes. The second was observed more frequently, was voltage-independent and fully blocked by saturating doses of nifedipine (10 M). Both actions were prevented by intracellular perfusion with guanosine 5-O-(2-thiodiphosphate) (GDP--S). Our data suggest that beside a majority of L-type channels, RINm5F and human pancreatic -cells may express a variable fraction of DHP-insensitive channels that may be involved in the control of insulin secretion during -cell activity.     

Tedisamil blocks single large-conductance Ca2+-activated K+ channels in membrane patches from smooth muscle cells of the guinea-pig portal vein

Enzymatically dispersed smooth muscle cells of the guinea-pig portal vein were studied by the patch-clamp technique. They were found to have Ca²⁺-dependent K⁺ channels with the typical properties of the BK channel, i.e. a reversal potential at the calculated equilibrium potential for K⁺ ions, a striking voltage dependence, and a conductance of approximately 200 pS ([K⁺]₀ 50 mM, [K⁺]_i 150 mM, positive patch potentials). Tedisamil, a new bradycardic agent with an inhibitory action on K⁺ currents in heart muscle, reduced the open probability of the BK channels concentration-dependently (1–100 M) when applied at the cytosolic side of membrane inside-out patches. At 100 M [Ca²⁺]_i, the IC₅₀ of tedisamil was 13.8 M (¯x, n=5). Tedisamil increased the frequency of channel closures, and reduced the mean duration of openings from 8 ms to < 1 ms, while the mean duration of closures within bursts (1–2 ms) was not altered. Tedisamil did not affect long closures (> 160 ms) between bursts, either. The mean time of residence of tedisamil at the BK channel was estimated to be 1–2ms. Hence, tedisamil, in comparison to the slow blocker Ba²⁺ and the fast blocker tetraethylammonium, holds the position of an intermediate K⁺ channel blocker.Supported by the Deutsche Forschungsgemeinschaft     

Inhibition of calmodulin expression prevents low-pH-induced gap junction uncoupling inXenopus oocytes

The relationship among intracellular pH (pH_i), –log₁₀ intracellular Ca²⁺ concentration (pCa_i) and gap junctional conductance, the participation of Ca²⁺ stores, and the role of calmodulin in channel regulation have been studied inXenopus oocytes, expressing the native connexin (Cx38), exposed to external solutions bubbled with 100% CO₂. The time courses of pH_i [measured with 2,7-bis(2-carboxyethyl)-5,6-carboxylluorscein (BCECF)], (pCa_i) (measured with the membrane-associated fura-C₁₈) and junctional conductance (measured with a double voltage-clamp protocol) were compared. The data obtained confirm previous evidence for a closer relationship of junctional conductance with (pCa_i) than with pH_i. Evidence for an inhibitory effect of intracellularly injected ruthenium red or 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) on CO₂-induced uncoupling, coupled to negative results with Ca²⁺-free external solutions, point to a low-pH_i-induced Ca²⁺ release from internal stores, likely to be primarily mitochondria. The hypothesis proposing a participation of calmodulin in channel gating was tested by studying the effects of calmodulin expression inhibition by intracellular injection of oligonucleotides antisense to the two calmodulin mRNAs expressed in the oocytes. Calmodulin mRNA was permanently eliminated in 5 h. The oocytes injected with the antisense nucleotides progressively lost the capacity to uncouple with CO₂ within 72 h. The effect of CO₂ on junctional conductance was reduced by 60% in 24 h, by 76% in 48 h and by 93% in 72 h. Oocytes that had lost gating sensitivity to CO₂. partially recovered gating competency following calmodulin injection. The data suggest that lowered pH_i uncouples gap junctions by a Ca²⁺-calmodulin-mediated mechanism.     

Neuron Activity in the Prefrontal Cortex of the Brain in Rats with Different Typological Characteristics in Conditions of Emotional Stimulation

Male Wistar rats were separated according to the emotional resonance method (groups of animals avoiding (altruists) and not avoiding (egotists) the pain cries of partner rats) and neuron activity in the prefrontal areas of the cortex was studied in the right and left hemispheres. Assessments were made of changes in the frequency of nerve cell spike activity (in relation to the baseline activity of neurons in sated animals) in rats subjected to one day of food deprivation and after electrical stimulation of emotionally positive (lateral hypothalamus) and negative (tegmentum of the midbrain) brain structures and after exposure to the pain cries of partner rats. The results of these experiments revealed a series of differences in the cell activities of the two groups of rats. In conditions of hunger, the discharge frequency in the altruists was higher than that in egotists. Cortical neuron responses to positive stimulation were greater than those to negative stimulation in rats of both groups. Intracerebral stimulation produced significantly greater increases in discharge frequency in neurons of both prefrontal areas of the cortex in altruists than in egotists. In both groups of rats, neurons in the right hemisphere responded to emotionally negative stimulation with significantly greater activation than cells in the left hemisphere, while activity in the left hemisphere was greater in conditions of emotionally positive stimulation. Altruists showed significantly greater neuron responses during exposure to pain cries from victim rats in both the right and left hemispheres. The responses of egotists to victim cries were not significantly different from baseline activity levels.