Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

The cation selectivity of the sarcoball Ca2+ channel in frog muscle fibres

 We have measured single-channel currents from sarcoplasmic reticulum (SR) blebs (sarcoballs) of frog skeletal muscle fibres using conventional patch-clamp electrodes with excised patches. With both the pipette and bath solutions containing 50 mM Ca(gluconate)₂ the slope conductance of the single channels was 39.2 pS for the most commonly seen state, with a reversal potential of –0.4 mV. The cation selectivity of this channel was investigated by replacing the bathing solution with either gluconate or HEPES salts of selected cations. The Goldman permeability ratios, calculated from the reversal potentials, were found to be P(Ca²⁺)/P(K⁺)=2.4, P(Ca²⁺)/ P(Na⁺)=2.7, P(Ca²⁺)/P(Tris⁺)=3.1, P(Ca²⁺)/P(Mg²⁺)=1.0 and P(Ca²⁺)/P(Ba²⁺)=1.1. Each value for the monovalent ions was found to be less than the corresponding value reported for the SR ryanodine receptor channel from skeletal and cardiac muscle. Single-channel activity could be recorded when the preparation was bathed in symmetrical 50 mM Mg(gluconate)₂ solutions, and these channels had a similar conductance and open probability to that measured when the preparation was bathed in symmetrical Ca(gluconate)₂ solution. The channel activity in symmetrical 50 mM Ca(gluconate)₂ solution was insensitive to bath-applied caffeine (5 mM) and ryanodine (10 μM). The results are in agreement with the conclusion that the sarcoball Ca²⁺ channel is not the ryanodine receptor release channel, but possibly a form of the SR Ca²⁺-ATPase which is uncoupled from the catalytic events of the pump and acts as a passive ion channel. Received: 13 February 1998 / Received after revision: 6 April 1998 / Accepted: 7 April 1998     

The role of cytosolic Ca2+ in the secretion of NaCl in isolated in vitro perfused rectal gland tubules of Squalus acanthias

 In many exocrine glands cytosolic Ca²⁺ ([Ca²⁺]_i) plays a pivotal role in stimulation-secretion coupling. In the rectal gland of the dogfish Squalus acanthias this appears not to be the case and it is believed that secretion is mainly controlled by the Cl^– conductance of the luminal membrane. We have examined this question in a study of isolated in vitro perfused rectal gland tubules (RGT). Three types of measurements were performed: (1) measurements of [Ca²⁺]_i by the fura-2 technique; (2) measurements of transepithelial electrical parameters, i.e. transepithelial voltage (V _te), transepithelial resistance (R _te), the equivalent short-circuit current (I _sc) and the voltage across the basolateral membrane (V _bl), and (3) whole-cell patch-clamp measurements of cellular voltage (V _m), conductance (G _m) and membrane capacitance (C _m). The data indicates that carbachol (CCH) increases [Ca²⁺]_i by increasing store release and Ca²⁺ influx. Other agonists, producing cytosolic cAMP, also increased [Ca²⁺] by enhancing Ca²⁺ influx. CCH hyperpolarized these cells and enhanced G _m significantly. The effect of CCH on V _te and I _sc was most marked under control conditions and disappeared in RGT otherwise stimulated by agonists that lead to cAMP production. It is concluded that [Ca²⁺]_i plays a major role in the stimulation of NaCl secretion in RGT by enhancing the basolateral K⁺ conductance. cAMP-producing agonists enhance [Ca²⁺]_i by increased Ca²⁺ influx. CCH releases Ca²⁺ from respective stores. CCH, unlike the cAMP-producing agonists, only increases basolateral K⁺ conductance. It modulates secretion especially under conditions in which the cAMP pathway is not fully activated. Received: 25 November 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

BK channels in intact clonal rat pituitary cells are activated by physiological elevations of the cytosolic Ca2+ concentration at the normal resting potential

Activation of large conductance Ca²⁺-activated K⁺ channels (BK channels) in intact clonal rat pituitary cells (GH₄ cells) was investigated using the cell-attached patch-clamp configuration. This method prevents loss of intracellular factors which might influence channel activity. BK channels are generally considered to be inactive at the resting membrane potential in excitable cells. However, at the resting potential (0 mV pipette potential), 40% of the cell-attached patches displayed spontaneously active BK channels, which remained active even at 20 mV hyperpolarization. The peptide thyroliberin (TRH) elevates the cytosolic Ca²⁺ concentration ([ Ca²⁺]_i) in GH cells by IP₃-induced release of Ca²⁺ from intracellular stores. This rise in [Ca²⁺]_i occurs concomitantly with membrane hyperpolarization. TRH stimulation caused activation of BK channels in nine out of 30 silent cell-attached patches, and caused enhanced channel activity in seven out of 29 cell-attached patches containing spontaneously active BK channels. The Ca²⁺ ionophore ionomycin activated silent BK channels in three out of 10 cell-attached patches, and increased the activity of spontaneously active BK channels in seven out of 16 cell-attached patches. The pipette potential was clamped to 0 mV in all these experiments. We conclude that the BK channels in GH₄ cells may be active at the resting membrane potential and more negative membrane potentials. The channels may also be activated further by physiological elevations of [Ca²⁺]_i in the same potential range. Our results point towards new possible physiological roles for the BK channels in GH₄ cells. This is in agreement with the emerging picture of BK channels highly sensitive to [Ca²⁺]_i in a wide variety of cell types.     

The effect of Ca2+ on the metarhodopsin I–II transition

Summary The effect of Ca²⁺ on kinetics and equilibrium of the Meta I–II transition was studied in rhodopsin-digitonin-solutions using flash-photometry. With increasing Ca²⁺-concentration the Meta I–II-equilibrium is shifted to Meta I. The pH-dependence of the Meta I–II equilibrium is suppressed by Ca²⁺. To obtain the same effect as with bivalent cations about the 10-fold concentration of univalent ions is required. Ca²⁺-ions have also an effect on the rate of equilibrating Meta I–II: with increasing Ca²⁺-concentration the rate-constants of the rapid and slow component decrease and become equal to the value at pH 8. This observation can be described as an inhibition of the catalytic effect of protons by Ca²⁺. Similar results are obtained with Mg²⁺, whereas K⁺ and Na⁺ are practically ineffective. In the presence of the Ca²⁺-blocking agents verapamil (Isoptin^®) and D-600 the rate of equilibrating Meta I–II is reduced. These and several former observations can be explained by a model in which the Meta I–II transition is coupled with the separation of negative fixed changes, which can be clamped by Ca²⁺.This work was supported by the Deutsche Forschungsgemeinschaft project No. Em 18/1-4     

α4β1 integrin promotes accumulation of tissue‐resident memory CD8+ T cells in salivary glands

The salivary glands (SGs) of virus‐immune mice contain substantial numbers of tissue‐resident memory CD8⁺ T cells (T_RM cells) that can provide immunity to local infections. Integrins regulate entry of activated T cells into nonlymphoid tissues but the molecules that mediate migration of virus‐specific CD8⁺ T cells to the SGs have not yet been defined. Here, we found that polyinosinic‐polycytidylic acid (poly(I:C)) strongly promoted the accumulation of P14 TCR‐transgenic CD8⁺ T_RM cells in SGs in an α₄β₁ integrin‐dependent manner. After infection with lymphocytic choriomeningitis virus, accumulation of P14 T_RM cells in SGs and intestine but not in kidney was also α₄ integrin dependent. Blockade of α₄β₇ by monoclonal antibodies (mAbs) inhibited lymphocytic choriomeningitis virus‐induced accumulation of P14 T_RM cells in the intestine but not in SGs. In conclusion, our data reveal that α₄β₁ integrin mediates CD8⁺ T_RM accumulation in SGs and that poly(I:C) can be used to direct activated CD8⁺ T cells to this organ.     

The effect of ouabain on intracellular activities of K+, Na+, Cl−, H+ and Ca2+ in proximal tubules of frog kidneys

Using conventional and ion selective microelectrodes, the effect of ouabain (10^–4 mol/l) on peritubular cell membrane potential (PD_pt), on intracellular pH (pH_i) as well as on the intracellular ion activities of Cl^– (Cl _i ^– ), K⁺ (K _i ⁺ ), Na⁺ (Na _i ⁺ ) and Ca²⁺ (Ca _i ²⁺ ) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PD_pt=–57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H⁺ {ie6-3}, of Na⁺ {ie6-4} and of Ca²⁺ {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PD_pt, which is reduced to half (PD_{pt, 1/2}) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PD_{pt, 1/2} apparent Cl _i ^– increases by 6.2±1.0 mmol/l, pH_i by 0.13±0.03, Ca _i ²⁺ by 185±21 nmol/l, and Na _i ⁺ by 34.2±4.6 mmol/l, whereas K _i ⁺ decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K⁺, by an accumulation of Ca²⁺, Na⁺ and HCO ₃ ^- in the cell and by a dissipation of the electrochemical Cl^– gradient.Supported by Österr. Forschungsrat, Proj. No. 4366     

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: Evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca²⁺ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokin-in-octapeptide (CCK₈) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av) in a suspension of acinar cells. However, in the presence of 1.0 M Staurosporine the stimulatory effect of submaximal concentrations of CCK₈ was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca²⁺]_i,av in cells prestimulated with a submaximal concentration of CCK₈. The data obtained with staurosporine indicate that CCK₈-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca²⁺ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK₈-induced Ca²⁺ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK₈-evoked increase in [Ca²⁺]_i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK₈ at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca²⁺]_i,av evoked by the CCK₈ analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca²⁺]_i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK₈ concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca²⁺]_i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca²⁺ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C. The data presented are in support of a negative-feedback role for protein kinase C in the process of receptor-mediated Ca²⁺ mobilization by a process that involves phosphorylation of the CCK receptor, thereby transforming it from a high-affinity state into a low-affinity state.     

Suppression of Ca2+ oscillations induced by cholecystokinin (CCK) and its analog OPE in rat pancreatic acinar cells by low-level protein kinase C activation without transition of the CCK receptor from a high- to low-affinity state

Cholecystokinin (CCK) analogs, JMV-180 and OPE, release Ca²⁺ from intracellular stores and induce oscillations in the concentration of cytosolic Ca²⁺ ([Ca²⁺]_i), but do not generate a detectable rise in inositol 1,4,5-trisphosphate (InsP ₃) levels. In contrast, high concentrations of CCK elevate InsP ₃, as well [Ca²⁺]_i, to a peak which decreases to near basal levels without oscillations. The mechanisms which underlie inhibition of [Ca²⁺]_i oscillations observed with high CCK concentrations are unclear, but are believed to involve a low-affinity CCK receptor state. Alternately, CCK analogs may be weak partial agonists of the phospholipase C pathway, whereas native CCK, as a full agonist of this pathway, stimulates low levels of protein kinase C (PKC) activity. Preincubation of acini with 1 nM 12 O-tetradecanoyl-phorbol 13-acetate (TPA) for 15 min at 37°C did not affect OPE binding to acini, but abolished OPE-induced (at 1 M) [Ca²⁺]_i oscillations without affecting the initial [Ca²⁺]_i spike. These transformed OPE-induced [Ca²⁺]_i responses mimicked those induced by supramaximal CCK octapeptide (CCK-8) concentrations. Inhibition of [Ca²⁺]_i oscillations by 1 nM TPA was reversed by the PKC inhibitor staurosporine (0.2 M). After [Ca²⁺]_i oscillations were induced with OPE or low concentrations of CCK-8 (20 pM), 1 nM TPA caused a gradual slowing of oscillation frequency over 15–20 min without affecting [Ca²⁺]_i spike amplitude. In contrast, 1 M TPA inhibited OPE binding and caused a more generalized inhibition of OPE- and CCK-evoked Ca²⁺ signals. These data suggest that inhibitory effects of low-level PKC activation on agonist-evoked Ca²⁺ signalling are distinct from the effects of high-level PKC activation by 1 M TPA, and do not require the transition of the CCK receptor from a high-affinity to a low-affinity state.     

Inflammatory level under different p53 mutation status and the regulation role of curcumin in tumor microenvironment

The inflammation is tightly associated with tumor development, promoting or inhibiting tumorigenesis. And mutant p53 is speculated to promote inflammation and tumorigenesis. The tumor associated macrophages are usually educated to present the anti-inflammatory profile to tune down antitumor immunity. However, the impact of p53 mutants on macrophages is not clear. Here, we compared the basal inflammatory level and macrophage profiles in tumor cells and tumor samples with different p53 mutations. Data revealed that a lower inflammatory level was maintained in immune organs and tumor cells with p53 point mutations than those with p53 null mutation. Using the tumor cell-derived conditional media to culture macrophages, we found that the media from cells with p53 mutations, especially the point mutations, could decrease M1 markers and inhibit phagocytosis, suggesting the p53 mutation promoted M2 profile polarization.To target the p53 mutation induced M2 macrophage polarization, we applied low-concentration curcumin to the tumor cells with different p53 mutations. The data showed that curcumin could inhibit STAT3 signal and decrease PPARγ and CSF1 in tumor cells and tumor samples. In vitro, the co-culture assays showed that the curcumin treatment shifted p53 mutation educated macrophages back towards M1 profile. In vivo, the curcumin-treated MEFs showed obvious tumor inhibition, and the tumor samples displayed inhibited M2 markers. Results suggested that curcumin could inhibit p53 mutation educated macrophage induction and suppress M2-promoted tumorigenesis.Our study illustrated the inflammatory level under different p53 status and the inflammatory regulated role of curcumin in tumor environment. This study might provide a potential method in tumor personalized treatment aiming immune therapy in different p53 status.