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.     

Functional adaptation to high PCO2 of apically and basolaterally located Na+/H+ exchange activities in cultured renal cell lines

Cultured renal epithelial cells grown on filter support were examined for functional adaptation of Na⁺/H⁺ exchange activities to respiratory acidaemia, which was mimicked by increasing PCO₂ from 5% to 10% during 24 h or 48 h of cell culture. We have selected proximal tubular cell lines with either dual location of Na⁺/H⁺ exchange activities (MCT cells, RKPC-2 cells), apical location of Na⁺/H⁺ exchange activity (OK/ WOK cells) or a basolateral location of Na⁺/H⁺ exchange activities (LLC-PK₁/clone 4 cells, MDCK cells). Na⁺/H⁺ exchange activity was determined microspectrofluorometrically (using BCECF) in the absence of CO₂/HCO ₃ ^– . Respiratory acidaemia specifically increased apical Na⁺/H⁺ exchange activity (previously classified as amiloride-resistant) in MCT cells, in RKPC-2 cells and in WOK cells; it stimulated basolateral Na⁺/H⁺ exchange activity (previously shown to be amiloride-sensitive) in RKPC-2 cells, in LLC-PK₁/clone 4 cells and in MDCK cells, but did not affect basolateral Na⁺/H⁺ exchange activity in MCT cells. In MCT and in RKPC-2 cells the effect of high PCO₂ on apical Na⁺/H⁺ exchange was prevented by inhibition of protein kinase C. In RKPC-2 cells, activation of basolateral Na⁺/H⁺ exchange by high PCO₂ occurred also when protein kinase C was inhibited. In conclusion, these studies demonstrate stimulation of apical Na⁺/H⁺ exchange, but differential regulation of basolateral Na⁺/H⁺ exchange activities in response to a high-PCO₂-induced acid environment. Protein kinase C activation might be involved in mediating the effect of acidaemia on stimulation of apical Na⁺/H⁺ exchange activity (MCT and RKPC-2 cells).     

Adrenergic and cholinergic inhibition of Ca2+ channels mediated by different GTP-binding proteins in rat sympathetic neurones

Effects of acetylcholine (ACh) and noradrenaline (NA) on voltage-gated ion channels of sympathetic neurones acutely dissociated from rat superior cervical ganglion (SCG) were examined using the whole-cell voltage-clamp technique. Depolarizing voltage steps elicited two types of low- and high-voltage-activated (LVA and HVA) Ca²⁺ currents. Pressure applications of ACh and NA produced concentration-dependent inhibition of the HVA Ca²⁺ current without affecting the LVA Ca²⁺ current. The inhibitory action of ACh on the Ca²⁺ current was blocked by a muscarinic antagonist, atropine. The action of NA was suppressed by an ₂-adrenergic antagonist, yohimbine, but not by an ₁-adrenergic antagonist, prazosin. Delayed rectifying outward K⁺ currents and inward rectifying K⁺ current were not affected by either ACh or NA. Tetrodotoxin-sensitive and -insensitive Na⁺ currents also remained unaffected under actions of ACh and NA. When recorded with electrode containing guanosine-5-O-(3-thiotriphosphate) (GTP--S), the inhibitory actions of ACh and NA on Ca²⁺ currents became irreversible. After treatment of SCG neurones with pertussis toxin, the inhibitory action of ACh on the Ca²⁺ current was almost completely abolished, whereas the action of NA was only partially reduced. The results suggest that ACh and NA differentially inhibit the HVA Ca²⁺ current via different G proteins coupling muscarinic and ₂-adrenergic receptors to Ca²⁺ channels in rat SCG neurones.     

Forskolin-induced block of delayed rectifying K+ channels in pancreaticβ-cells is not mediated by cAMP

K⁺ channels in the membrane of murine pancreatic -cells were studied using the patch-clamp technique. The delayed outward current was activated in whole-cell experiments by depolarizing voltage pulses to potentials between –30 mV and 0 mV. Forskolin blocked the current rapidly (<5 s) and reversibly with 50% inhibition at 13 M. The inhibition did not depend on a stimulation of the adenylate cyclase since it occurred even in presence of 1 mM cAMP in the pipette solution which replaced the cytoplasm. Membrane permeant cAMP analogues and phosphodiesterase inhibitors did not influence the delayed outward current. In experiments on outside-out patches forskolin (100 M) shortened the openings of a channel of about 10 pS conductance at 0 mV and a time course of activation and inactivation similar to the whole-cell current. Another smaller, slowly activating channel and the Ca²⁺- and ATP-dependent K⁺ channels were influenced only weakly or not at all. It is therefore concluded that the 10-pS channel generates most of the delayed outward K⁺ current in murine pancreatic -cells. The Ca²⁺-independent part of the delayed outward current in bovine adrenal chromaffin cells was also blocked by forskolin (100 M).     

Contractile protein gene expression in serum-free cultured adult rat cardiac myocytes

The effects of two adhesion substrates (serum and laminin) and time in culture on the expression of genes encoding myosin heavy chain (MHC) isoforms and -skeletal actin were analysed in myocytes isolated from adult rat heart and maintained in serum-free culture. Relative messenger ribonucleic acid (mRNA) abundances were quantitated by dot-blot analysis. Gene expression was not influenced by the substrate used. Time in culture induced a decrease in total mRNA abundance and an up-regulation of -MHC and -skeletal actin genes. It is proposed that atrophy of adult myocytes is associated with a pattern of gene expression similar to the fetal program.     

Liberation ofβ-lipoprotein,alkaline phosphatase,and 5′-nucleotidase from platelets in response to aggregation induced by adrenalin

During aggregation induced by adrenalin,-lipoprotein, alkaline phosphatase, 5-nucleotidase, and factor 3 are liberated from the platelets. The electrophoretic mobility of platelet alkaline phosphatase is the same as that of the-lipoprotein. This suggests that the-lipoprotein, alkaline phosphatase, and 5-nucleotidase are structural components of platelet material carrying the factor 3 activity.Department of Biochemistry, S. V. Kurashov Medical Institute, Kazan'. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No, 5, pp. 545–547, May, 1976     

Development of traumatic shock accompanied by increased activity of polyspecific antibodies reacting with DNA and endotoxins

Department of Radiation Biochemistry, Research Institute of Medical Radiology, Academy of Medical Sciences of the USSR, Obninsk. Central Research Laboratory, Rostov Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Nasonova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 5, pp. 538–539, May, 1991.     

Rapid refilling of Ca2+ stores in macrophages stimulated by ATP involves the sequential activation of phospholipase D and protein kinase C

Ca²⁺ movements between intracellular stores, the cytoplasm and external solution were analysed in murine peritoneal macrophages stimulated by various agonists. The Ca²⁺ content of intracellular stores was estimated from the amplitude of Ca²⁺-transients elicited by ionomycin applied in Ca²⁺-free solution. Both uridine 5-triphosphate (UTP) and platelet-activating factor (PAF) triggered the release of Ca²⁺ followed by a sustained influx, during which intracellular stores remained totally empty. In contrast, in the continuous presence of adenosine 5-triphosphate (ATP), Ca²⁺ was initially released and then rapidly sequestered again by the stores. ATP-induced store refilling was not related to cell depolarization or to an increase in the intracellular Na⁺ concentration (two specific consequences of ATP stimulation which are not induced by PAF and UTP). Store refilling was not caused by a signal that ATP would fail to induce (e.g. as a result of receptor desensitization), but was positively controlled by ATP, even in the simultaneous presence of a concentration of PAF which, on its own, would have caused a persistent store depletion. The hypothesis that the signal delivered by ATP involves the sequential activation of phospholipase D and protein kinase C is consistent with the present pharmacological evidence. However, although we found conditions in which Ca²⁺ stores did not refill in the presence of ATP, this maintained store depletion was not accompanied by a sustained Ca²⁺ response similar to that elicited by PAF or UTP, suggesting that store depletion is a condition which is necessary, but not sufficient, for inducing Ca²⁺ influx.     

Role of adrenergic structures in functional control over the cerebral circulation

An investigation of the cerebral circulation by the thermoelectric method showed that stimulation of the cervical sympathetic nerve leads to considerable changes in the blood supply to the brain. The changes in blood flow are biphasic in character: An initial small increase is followed by a decrease below the original level. Pharmacological analysis with and adrenoblockers showed that the constrictor response of the cerebral vessels is due to excitation of-adrenergic structures and the dilator response to excitation of-adrenergic structures. A possible mechanism of these changes is postulated.Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 9–12, January, 1976.     

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.     

The isolated frog skin epithelium: Presence of α and β adrenergic receptors regulating active sodium transport and water permeability

Summary The effects of stimulation of and adrenergic receptors on short circuit current (S.C.C.), Na⁺ and Cl^– fluxes and osmotic water permeability were studied on isolated frog skin epithelial layers separated from the dermis.Low norepinephrine doses (final concentrations in the incubation medium ranging from 5×10^–9 to 10^–8 M) produced increased water permeability and S.C.C. The latter was entirely accounted for by an increase in the active Na⁺ influx. Na⁺ outflux and Cl^– fluxes were not modified. Both these effects disappeared after treatment with the blocking agent, Propranolol. Higher norepinephrine doses (final concentrations: 10^–7 to 10^–6 M) produced: 1. an increase in water permeability lower than that produced by low doses, the highest doses failing to increase water permeability, and 2. a triphasic change in S.C.C.: after an initial increase, S.C.C. dropped to its resting value and then rose again to a sustained value. Na⁺ and Cl^– flux measurements showed that the variation in S.C.C. reflected variations in active Na⁺ transport. When the same high norepinephrine doses were applied after treatment with the blocking agent Phentolamine, the effects observed were identical to those obtained with low doses.On blocked preparations, large doses of norepinephrine inhibited the water permeability and sodium transport increases induced by theophylline or oxytocin but did not modify those induced by 35-cyclic AMP. The inhibition was suppressed after blocking receptors.From the foregoing, it was concluded that both and adrenergic receptors are present in frog skin epithelial cells and are involved in the regulation of water and sodium permeability.It is suggested that the inhibitory effect of stimulation resulted from the inhibition of cyclic-AMP generating system, the activity of which is under the positive control effect of oxytocin and stimulation.     

Modulation of Ca2+-activated K+ channels by Mg2+ and ATP in frog oxyntic cells

Ca²⁺-activated K⁺ channels in the basolateral plasma membrane of bullfrog oxynticopeptic cells are intimately involved in the regulation of acid secretion. Patch-clamp techniques were applied to study the regulating mechanism of these channels. In the excised inside-out configuration, intracellular Mg²⁺ decreased channel activity in a dose-dependent manner. In the absence of Mg²⁺, administration of adenosine 5 triphosphate (ATP) to the cytoplasmic side also inhibited channel activity. On the other hand, in the presence of Mg²⁺, addition of ATP markedly increased channel activity. At a fixed concentration of free Mg²⁺ the Mg-ATP complex caused channel activation and shifted the dose response relationship between channel activity and the intracellular Ca²⁺ concentration to the left. A nonhydrolysable ATP analogue, adenosine 5-[,-imido]triphosphate (AMP-PNP) adenylyl [,-methylene]diphosphate (AMP-PCP), could not substitute for ATP in channel activation, but a hydrolysable ATP analogue, adenosine 5-O-(3-thiotriphosphate) (ATP[S]) could do so. Furthermore, application of alkaline phosphatase to the cytoplasmic side inhibited channel activity. These results demonstrate that Ca²⁺-activated K⁺ channels are regulated by Mg²⁺ and ATP, and suggest that a phosphorylation reaction may be involved in the regulation mechanism of these channels.     

Role of adrenergic structures in functional control over the cerebral circulation

An investigation of the cerebral circulation by the thermoelectric method showed that stimulation of the cervical sympathetic nerve leads to considerable changes in the blood supply to the brain. The changes in blood flow are biphasic in character: An initial small increase is followed by a decrease below the original level. Pharmacological analysis with and adrenoblockers showed that the constrictor response of the cerebral vessels is due to excitation of -adrenergic structures and the dilator response to excitation of -adrenergic structures. A possible mechanism of these changes is postulated.Laboratory of Pathophysiology of Respiration, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 9–12, January, 1976.     

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.     

Control of metastatic properties of BL6 melanoma cells by H-2Kb gene: immunological and nonimmunological mechanisms

The effect of class I H-2 antigen expression on the metastatic properties of BL6 melanoma cells was investigated. The BL6-8 clone isolated from the highly metastatic BL6 melanoma did not express H-2K b gene. Following transfection with the H-2K^b gene, BL6-8 cells displayed a low metastatic potential in the immunocompetent as well as immunosuppressed (X-irradiated) or triple-immunodeficient mice with impaired T, B and natural killer (NK) cells function. The expression of H-2K^b gene and the low metastatic ability of transfected BL6 melanoma cells were associated with appearance of cell membrane soybean agglutinin (SBA) and Griffonia simplicifolia 1B₄ (GS1B₄) lectin-binding carbohydrataes. These alterations in cell surface carbohydrates were found to be a result of reduction in sialylation of SBA binding sites and upregulation of the 1.3 galactosyltransferase (1.3GT) gene. To assess the importance of H-2K^b-induced alterations in cell surface carbohydrates for metastasis formation, BL6-8 melanoma cells were transfected with H-2K^b gene without neo^r gene cotransfection and selected for adherence to SBA-lectin-conjugated agarose beads. The transfected clones that expressed SBA and GS1B₄ lectin-binding carbohydrates were low metastatic. Further analysis of these clones showed that presence of SBA and GS1B₄ lectin-binding carbohydrates rather than expression of H-2K^b molecules per se might be responsible for low metastatic potentials of H-2K^b-transfected cells in the immunocompromized mice. Studies of the possible mechanisms responsible for low metastatic ability of H-2K^b-transfected melanoma cells revealed that these cells displayed a reduced ability to adhere to murine pulmonary endothelial cells as well as to laminin and collagen IV. We hypothesized that the observed nonimmunological effects of H-2K^b gene in BL6 melanoma cells is a result of an interaction between the H-2K^b gene and B16 melanoma-specific ecotropic retrovirus. It results in inhibition of this retrovirus production with consecutive alteration in the expression of cellular genes controlling cell surface glycosylation and adhesion properties essential for the metastatic phenotype of BL6 melanoma.     

Nitric oxide mediated modulation of free radical generation response in the rat polymorphonuclear leukocytes: A flowcytometric study

Nitric oxide (NO) synthesis and free radical generation from polymorphonuclear leukocytes (PMNs) play an important role in several pathological conditions. It is therefore important to understand the regulatory mechanisms of free radical generation from PMNs. Flowcytometry can be used to assess generation of reactive oxygen and nitrogen species from PMNs by using fluorescent probes. In the present study regulation of NO synthesis in the control and lipopolysaccharide (LPS) treated rat PMNs has been investigated. Free radical generation was assessed by flow cytometry using a dye, 27-dichlorodihydrofluorescein diacetate (DCFDA), dihydrorhodamine-123 (DHR) and 4,5-diaminofluorescein diacetate (DAF). Superoxide dismutase (SOD), and catalase significantly attenuated the arachidonic acid (AA, 1 × 10^–6 M) induced free radical generation, while 4-aminobenzoicacid hydrazide (ABH), myeloperoxidase (MPO) inhibitor had no significant effect. Intracellular and extracellular calcium levels also modulated FR generation. AA induced free radical generation from PMNs was also enhanced significantly after LPS treatment. NO synthase (NOS) inhibitors, aminoguanidine (AG) and 7-nitroindazole (NI) inhibited arachidonic acid induced free radical generation from LPS treated PMNs, while in control PMNs NOS inhibition had no effect. Augmentation of free radical generation from rat PMNs following LPS treatment seems to be regulated by NO.