Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

Muscarinic modulation of calcium dependent plateau potentials in rat neostriatal neurons

Intracellular recording from neostriatal neurons in rat brain slices revealed effects of the acetylcholine (ACh) agonist carbachol (Cch, 1–10 mol/l), of the anticholinesterase physiostigmine (10 mol/l) and of the muscarinic antagonist atropine (10 mol/l) on plateau potentials elicited in the presence of K-blockers were Cadependent, elicited in the presence of K-blockers were Cadependent, since they persisted in Na-free solution, were resistant to tetrodotoxin (TTX, 3 mol/l) and blocked by Cd (0.1–0.5 mmol/l). Cch reduced the duration of the plateau potentials and made them more susceptible to fatigue. These effects were antagonized by atropine (1–10 mol/l), but not by Ba (100–200 mol/l) or 4-aminopyridine (4-AP, 0.5 mmol/l). Physostigmine (10 mol/l) had the same atropine-sensitive effects as Cch on the plateau potential. Atropine (10 mol/l), by itself, prolonged the duration of the plateau potential. High concentrations (100 mol/l) of Cch did not further reduce the duration of the plateau potential, instead, the duration re-increased with prolonged exposure. The re-increase of the plateau-spike duration was later masked by bursting activity. The opposing effects of low and high concentrations of Cch on the plateau potential duration corresponded to effects of this drug on intrastriatally evoked EPSPs in that low concentrations of Cch reduced the EPSP amplitude, but high concentrations re-increased it after a transient decrease. It is concluded that the muscarinic effect of Ach in the neostriatum is to modulate Ca-influx and that this effect is exerted in a tonic manner. On leave from absence from: Clinica Neurologica II, Universita di Roma. Tor Vergata, Roma, Italy.     

Inhibition of cysteinyl-leukotriene production by azelastine and its biological significance

Azelastine is a phthalazinone derivative with a wide spectrum of pharmacological activities. Actively sensitized guinea pigs were used to examine the broncholytic effect of azelastinein vivo. Furthermore, the influence of azelastine on the production of arachidonic acid (AA) metabolites was investigatedin vitro and compared to the effects of nordihydroguaiaretic acid (NDGA), indomethacin and ketotifen.In vivo, azelastine protected actively sensitized guinea-pigs against ovalbumin-induced bronchospasm with an ID₅₀ of 0.08 mg/kg orally. Ketotifen was similarly active (ID₅₀=0.05 mg/kg). Antigen-induced contraction of isolated tracheal rings of sensitized guinea-pigs was concentration-dependently inhibited by azelastine and NDGA with IC₅₀-values of 94.1 and 34.2 mol/l, respectively. Ketotifen exerted only weak inhibitory activity (18% at 100 mol/l). The arachidonic acid-induced contraction of isolated guinea-pig tracheal rings was also inhibited both by azelastine (IC₅₀=92.6 mol/l) and NDGA (IC₅₀=20.4 mol/l). Ketotifen was inactive on this model. Antigen challenge of chopped lung tissue from sensitized guinea-pigs resulted in the release of cysteinyl-leukotrienes (LT) which were identified by reversed phase high pressure liquid chromatography (HPLC) as LTD₄ and LTE₄. The release of cysteinyl-LT from sensitized guinea-pig lung tissue induced by antigen challenge was concentration-dependently inhibited by azelastine (IC₅₀=35.2 mol/l) and NDGA (IC₅₀=8.4 mol/l) but not by ketotifen and indomethacin. By contrast, indomethacin caused a pronounced augmentation of cysteinyl-LT release. The concentration of indomethacin, which augmented cysteinyl-LT release by 50% was 0.19 mol/l. At the same concentration, indomethacin inhibited the release of 6-keto-PGF₁ and TXB₂ by about 50%. Azelastine negligible influenced 6-keto-PGF₁ and slightly diminished TXB₂ release from the chopped lung tissue after challenge. Its IC₅₀-values were >2 mmol/l and 443 mol/l, respectively. NDGA inhibited the release of 6-keto-PGF₁ and TXB₂ with IC₅₀-values of 47.3 and 38.3 mol/l, respectively. Ketotifen was ineffective in inhibiting the release of cyclo-oxygenase products of AA metabolism. It seems likely that inhibition of release of 5-lipoxygenase-derived products of AA metabolism by azelastine contributes to its antiallergic and antiasthmatic activity.Author for correspondence.     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibers

The histamine-receptor-subtype-mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibers were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibers, histamine (3 mol/l) increased the action potential plateau height, decreased the action potential duration measured at a repolarization level of –60 mV and enhanced the pacemaker activity. These effects were abolished by the H₂-selective antagonist cimetidine (30 mol/l), but were not impaired by the H₁-selective antagonist dimetindene (0.3 mol/l).In spontaneously active Purkinje fibers, histamine (10 mol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole by 32% of the respective control measurements. These effects were blocked by 30 mol/l cimetidine, but remained unchanged in the presence of 0.3 mol/l dimetindene.Concentration-response curves of histamine were shifted to the right by approximately 2 logarithmic units in the presence of 30 mol/l cimetidine, but were not influenced in the presence of 0.3 mol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 mol/l) had similar actions as histamine on spontaneously active Purkinje fibers, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibers is exclusively mediated by H₂ receptors.Dedicated to Prof. Dr. E. Mutschler on the occasion of his 60th birthday.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr. 40008786.     

Actin-dependent activation of ion conductances in bronchial epithelial cells

Activation of Cl^– and K⁺ channels is necessary to drive ion secretion in epithelia. There is substantial evidence from previous reports that vesicular transport and exocytosis are involved in the regulation of ion channels. In the present study we examined the role of cytoskeletal elements and components of intracellular vesicle transport on ion channel activation in bronchial epithelial cells. To this end, cells were incubated with a number of different compounds which interact with either microtubules or actin microfilaments, or which interfere with vesicle transport in the Golgi apparatus. The effectiveness of these agents was verified by fluorescence staining of cellular microtubules and actin. The function was examined in ³⁶Cl^– efflux studies as well as in whole-cell (WC) patch-clamp and cell-attached studies. The cells were studied under control conditions and after exposure to (in mmol/l) ATP (0.1), forskolin (0.01), histamine (0.01) and hypotonic bath solution (HBS, NaCl 72.5). In untreated control cells, ATP primarily activated a K⁺ conductance whilst histamine and forskolin induced mainly a Cl^– conductance. HBS activated both K⁺ and Cl^– conductances. Incubation of the cells with brefeldin A (up to 100 mol/l) did not inhibit WC current activation and ³⁶Cl^– efflux. Nocodazole (up to 170 mol/l) reduced the ATP-induced WC current, and mevastatin (up to 100 mol/l) the cell-swelling-induced WC current. Neither had any effect on the WC current induced by forskolin and histamine. Also ³⁶Cl^– efflux induced by HBS, ATP, forskolin and histamine was unaltered by these compounds. Similarly, colchicine (10 mol/l) and taxol (6 mol/l) affected neither ³⁶Cl^– efflux nor WC current induced by ATP, forskolin, histamine or HBS. In contrast, depolymerisation of actin by cytochalasin D (10 mol/l) significantly attenuated ³⁶Cl^– effluxes and WC current activation by the above-mentioned agonists. Incubation with a C2 clostridial toxin (5 nmol/l) showed similar effects on WC currents. Moreover, when cytochalasin D (10 mol/l), C2 clostridial toxins (5 nmol/l), or phalloidin (10 mol/l) were added to the pipette filling solution current activation was markedly reduced. However, in excised inside-out membrane patches, cytochalasin D (10 mol/l), G-actin (10 mol/l) and phalloidin (10 mol/l) had no effect. These data suggest that actin participates in the activation of ion channels in 16HBE14o- epithelial cells and support the concept that exocytosis is a crucial step in the regulation of Cl^– and K⁺ channels in these cells.     

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.     

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37°C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was –50±0.4 mV (n=53). Cromakalim (BRL 34915), 1 mol/l and 10 mol/l, hyperpolarized the membrane potential by 9±1 mV (n=11) and 15±1 mV (n=53) respectively. Glibenclamide (10 mol/l) abolished the hyperpolarizing effect of cromakalim (n=6). Simultaneous addition of cromakalim and glibenclamide (both 10 mol/l, n=11) and glibenclamide itself (10 mol/l, n=7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca²⁺-sensitive K⁺ channels, cromakalim increased the open probability (P _o) only slightly and only with a cytosolic Ca²⁺ activity of 1 mol/l. In all other series cromakalim had no effect on the P _o of these channels. Forskolin (10 mol/l) hyperpolarized PD by 6±1 mV (n=13). The nucleotides UTP, ATP and ITP (10 mol/l) depolarized PD by 12±1 mV (n=7), 8±1 mV (n=65) and 5±1 mV (n=6) respectively. GTP, [,-methylene]ATP and adenosine had no significant effect. Mn²⁺ (1 mmol/l, n=18), Ni²⁺ (1 mmol/l, n=13), Co²⁺ (1 mmol/l, n=11), Zn²⁺ (1 mmol/l, n=6) and the Ca²⁺-channel blockers verapamil and nifedipine (both 0.1 mmol/l, n=6) did not attenuate the depolarization induced by 10 mol/l ATP. Fetal calf serum (100 ml/l, n=7) depolarized PD by 11±2 mV. This effect was not abolished by nifedipine or by replacing NaCl by choline chloride. The data indicate that PD of vascular smooth muscle cells is depolarized by P₂ agonists and hyperpolarized by the K⁺-channel opener cromakalim. The effect of cromakalim is antagonized by glibenclamide. The effect of cromakalim is probably not mediated by the K⁺ channel identified in excised patches.Supported by DFG Gr 480/10     

Effects of serotonin on electrical properties of Madin-Darby canine kidney cells

The present study has been performed to test for the influence of serotonin on the potential difference across the cell membrane (PD) of Madin-Darby canine kidney (MDCK)-cells. Under control conditions PD averages –48.6±0.6 mV (n=98). Increasing extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarizes the cell membrane by +6.3±0.6 mV (n=6) and +14.1±1.0 mV (n=12), respectively. The cell membrane is transiently hyperpolarized to –67.8±0.8 mV (n=63) by 1 mol/l serotonin. In the presence of serotonin, increasing extracellular potassium concentration from 5.4 to 20 mmol/l depolarizes the cell membrane by +26.4±1.0 mV (n=11). 1 mmol/l barium depolarizes the cell membrane by +15.7±1.3 mV (n=17) and abolishes the effect of step increases of extracellular potassium concentration from 5.4 to 10 mmol/l. In the presence of barium, serotonin leads to a transient hyperpolarization by –26.3±1.0 mV (n=16). During this transient hyperpolarization, the cell membrane is sensitive to extracellular potassium concentration despite the continued presence of barium. 10 mol/l methysergide hyperpolarize the cell membrane by –7.2±2.0 mV (n=6). In the presence of 10mol/l methysergide, the effect of serotonin is virtually abolished (+0.4±0.9 mV,n=6). 1 mol/l ketanserin, a 5-HT₂ receptor blocking agent, ICS 205-930, a 5-HT₃ receptor blocking agent, and phentolamine, an unspecific -receptor blocking agent, do not significantly modify the effect of serotonin. In the nominal absence of extracellular calcium, the effect of serotonin is markedly reduced. In conclusion, serotonin hyperpolarizes MDCK-cells by increasing apparent potassium conductance. This effect is transmitted by 5-HT₁ receptors and depends on extracellular calcium.     

Effects of PAF and PAF antagonists on the shape of venous endothelial cellsin vitro

Three platelet-activating factor (PAF) antagonists were tested for their ability to prevent or reduce PAF-induced shape changes of large vein endothelial cellsin vitro. BN52021 had a significant protective action at concentrations of 1 M and 0.1 M, but at 100 M had a damaging effect of its own. CV3988 (0.1 M and 1 M) and L652, 731 (20 M) did not reduce the responses to PAF, and at higher concentrations (CV3988 10 M and 100 M, L652, 731 100 M) both compounds alone caused significant changes of shape. BN52021 (0.1 M) was also effective against leukotriene (LT) C₄, at 1 M against bradykinin and LTE₄, and at 10 M against LTD₄ and the calcium ionophore A23187. BN52021 (10 M) was ineffective against shape changes induced by histamine, prostaglandin (PG) E₂ and lysophosphatidylcholine (LPC). Neither indomethacin (100 M) nor verapamil (20 M) altered the response to PAF.Using electron spin resonance (ESR) spectrometry it was shown that the damaging effects of LPC and CV3988 may be due partly to their detergent properties. It is suggested that the mechanism by which PAF alters the shape of large vein endothelial cells is primarily receptor mediated.     

The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation

We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (I _Cl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation ofI _Cl,vol occurred at a hypotonicity of 27.5 ± 1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (–)-indolactam did not affectI _Cl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 mol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19–31 pseudosubstrate peptide, did not influenceI _Cl,vol. Trifluoperazine and tamoxifen fully blockedI _cCl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 mol/1 respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca²⁺] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 ol/1) and genistein (100 ol/1) did not affectI _Cl,vol Exposing CPAE cells to lysophosphatidic acid (1mol/1), an activator of p42 MAPkinase and the focal adhesion kinase p125^FAK in endothelial cells, neither evoked a Cl^– current nor affectedI _Cl,vol Neither wortmannin (10 mol/1), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/1), which interferes with the p70S6 kinase pathway, affectedI _Cl,vol Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl^– current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl^– current induced by hypotonicity.     

Characterization of the Cl− conductance in the granular duct cells of mouse mandibular glands

We have previously shown that mouse mandibular granular ducts contain a hyperpolarization-activated Cl^– conductance. We now show that the instantaneous current/voltage (I/V) relation of this Cl^– conductance is inwardly rectifying with a slope conductance of 15.4±1.8 nS (n=4) at negative potentials and of 6.7±0.9 nS (n=4) at positive potentials. Thus, the inward rectification seen in the steady-state I/V relation is due, not only to voltage activation of the Cl^– conductance, but also to the intrinsic conductance properties of the channel. We show further that the ductal Cl^– conductance is not activated by including ATP (10 mmol/l) in the pipette solution. Finally, we show that the conductance is not blocked by the addition of any of the following compounds to the extracellular solution: anthracene-9-carboxylate (A9C, 1 mmol/l), diphenylamine-2-carboxylate (DPC, 1 mmol/l), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 mol/l), 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS, 100 mol/l), indanyloxyacetic acid (IAA-94, 100 mol/l), verapamil (100 mol/l), glibenclamide (100 mol/l) and Ba²⁺ (5 mmol/l). The properties of the ductal Cl^– conductance most nearly resemble those of the ClC-2 channel. Both channel types have instantaneous I/V relations that are slightly inwardly rectifying, are activated by hyperpolarization with a time-course in the order of hundreds of milliseconds, have a selectivity sequence of Br^–>Cl^–>I^–, and are insensitive to DIDS. The only identified difference between the two is that the ClC-2 channel is 50% blocked both by DPC and A9C (1 mmol/l), whereas the ductal Cl^– conductance is insensitive to these compounds.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.     

Atrial natriuresis under the condition of a constant renal perfusion pressure

An experimental elevation of left atrial pressure (eLAP ) by means of a reversible mitral stenosis is accompanied with an increase in sodium excretion (UNa—) and arterial blood pressure (by about 20 mm Hg, 2.7 kPa), and by a decrease in plasma renin activity.It is well established that an increase in renal perfusion pressure (Pren) can augment UNa—. Therefore the present study was undertaken to examine whether the eLAP -induced natriuresis was caused by the increased Pren. — Four female beagle dogs were kept under controlled environmental conditions. They received asodium rich diet (14.5 mmol/Na/kg/d). The dogs were chronically instrumented: purse string around the mitral annulus, catheter in the left atrium, carotid loop, pneumatic cuff above the renal arteries, pressure transducer below the renal arteries. Pren was kept constant by means of a digital servofeedback control circuit. The dogs served as their own controls (13 experiments without and 15 experiments with a controlled renal perfusion pressure were performed).After eLAP(+1.0 kPa), UNa— rose from 4.1±2.6 to 10.3±3.9 mol Na/min/kg. If Pren was kept constant, the corresponding values were 4.2±2.8 and 9.3±2.9 mol/min/kg. These data clearly indicate that the atrial natriuresis is not mediated by an augmentation of renal perfusion pressure. Therefore these results support the hypothesis that atrial natriuresis probably is due to an eLAP-induced suppression of the renin-angiotensin-system or other natriuretic mechanisms.Abbreviations ADP 3×20 min After distension period - AN Atrial natriuresis - bw kg Body weight - CP 3×20 min Control period - DP 3×20 min Distension period - eLAP kPa Experimental increase of left atrial pressure (during all DP's about +1.0 kPa) - HR l/min Heart rate - LAP kPa Left atrial pressure - n ₁ Number of dogs used - n ₂ Number of experiments (1 expt/day) - n ₃ Number of collection periods (urine) or number of samples (HR) - Part kPa Mean systemic arterial blood pressure (carotid artery) - Pren kPa Mean renal perfusion pressure (aorta, below the renal arteries) - UCI— mol/min/kg Chloride excretion - UK— mol/min/kg Potassium excretion - UNa— mol/min/kg Sodium excretion - Uosm— osm/min/kg Osmolar excretion - — l/min/kg Urine volume Preliminary parts of this work have been presented in Kiel (Spring meeting of the Deutsche Physiologische Gesellschaft, March 1979) and in Stockholm, Sweden (III. European Colloquium on Renal Physiology, June 1979).     

Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current

The effect of tyrosine protein kinase inhibitors on the swelling-induced chloride current (I _Cl-swelling of dog atrial myocytes was studied using the whole-cell patch-clamp recording technique. Currents were measured during hyperpolarizing voltage ramps with potassium currents blocked by cesium. Osmolarity was varied using mannitol. Exposure to hypotonic solution (249 mosmol/kg) activated I _Cl-swelling. Hypertonic solution ( 363mosmol/kg) was used to shrink swollen cells and turn off I _Cl-swelling. In studies on the acute effect of tyrosine protein kinase inhibitors each cell was swollen three separate times. Control, treatment, and washout I _Cl-swelling were compared. Genistein (50–80 M) prevented reactivation of I _Cl-swelling without affecting cell size. The effect of genistein partially subsided upon washout. The effect of genistein on I _Cl-swelling was not mimicked by 80 M daidzein, a related compound that does not inhibit tyrosine protein kinases. When intracellular adenosine 5-0-(3-thiotriphosphate (ATP[S]) was used, genistein did not prevent the reactivation of I _Cl-swelling. Intracellular ATP[S] did not result in a persistent activation of I _Cl-swelling when cell size was returned to control. Acute exposure to 1 M herbimycin A or 100 M tyrphostin 51 did not prohibit the activation of I _Cl-swelling. A 24-h exposure to 1 M herbimycin A did inhibit I _Cl-swelling. The results provide important clues regarding the activation mechanism for I _Cl-swelling and suggest that a tyrosine protein phosphorylation may be necessary, but not sufficient, for activation of I _Cl-swelling.     

Histamine H3-receptor activation inhibits acetylcholine release from the guinea pig myenteric plexus

The role of histamine H₃-receptors in the control of acetylcholine release from peripheral cholinergic neurons was evaluated in the isolated guinea pig ileum, previously loaded with³H-choline. When tested in the presence of H₁- and H₂-blockade, histamine (0.1–100 mol/l) and (R)-methylhistamine (0.01–1 mol/l) dose-dependently reduced the electrically-evoked choline outflow, with (R)-methylhistamine being a partial agonist. Selective H₃-receptor blocking drugs, thioperamide (0.1 mol/l) and impromidine (0.1 mol/l) reversed the histamine-induced inhibitory, effect. These data suggest that intestinal cholinergic nerves are endowed with histamine H₃-receptors whose activation produces an inhibitory effect upon acetylcholine release. The practical implications of these findings are obvious.     

The effects of indomethacin and verapamil on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Histamine (300 M), bradykinin (2 M), prostaglandin E₂ (PGE₂) (30 M), or the leukotrienes (LT) C₄ and E₄ (1 M) but not D₄ (1 M) appliedin vitro have been shown to change the shape of endothelial cells lining the guinea pig isolated thoracic inferior vena cava. All caused the formation of inter-endothelial cell gaps. Pre-treatment with either indomethacin (100 M) or verapamil (20 M) reduced the effects of these compounds. It is suggested that indomethacin and verapamil act by reducing the amount of intracellular calcium available for the shortening of contractile protein filaments within endothelial cells.     

Effect of a series of 1-alkyl ether lipids on inhibition of phospholipase A2 activity and PAF responses

Several 1-alkyl ether lipids were studied for their ability to inhibit PLA₂ and antagonize PAF responses. Studies with synthetic micellar substrate (1-stearyl-2-arachidonyl phosphocholine), at concentrations ranging from 0.02 to 1000M, demonstrate that CL 118326 inhibits porcine pancreatic PLA₂ in vitro. As the substrate concentration increases, there is a dose-dependent increase in the IC₅₀ value (IC₅₀ ranges: 1.6–84.6g/ml or 2.6–137M). CL 118326 inhibits mammalian pancreatic PLA₂, but not snake or bee venom PLA₂. CL 118326 inhibits thrombin (IC₅₀ =7.9M), but not Na arachidonate- (IC₅₀ > 100M) induced platelet aggregation, indicative of inhibition of cellular PLA₂. CL 118326 inhibits other PLA₂-dependent processes such as antigen-induced leukotriene (LTC₄) release (IC₅₀=2.3g/ml or 3.8M) and histamine release (IC₅₀=1.4g/ml or 2.2M) in basophil-enriched WBCs. Intradermal coinjection of CL 118326 (10g) with PLA₂ into guinea pig skin inhibits pancreatic PLA₂-induced increase in vascular permeability and leakage, but not snake or bee venom PLA₂-induced leakage. CL 118326 shows no PAF-like agonist activity in stimulating rabbit platelet-rich plasma. It inhibits PAF-induced aggregation (IC₅₀=5.8M), but not ADP-induced aggregation. CL 118326 has greater efficacy as a PLA₂ inhibitor than as a PAF antagonist since the IC₅₀-substrate concentration ratio for PLA₂ inhibition is <- 1.0 at substrate concentrations of 10–1000M while the IC₅₀-agonist ratio for PAF antagonism is > 100. Results for four other compounds related to CL 118326 are also presented.     

Veratridine-induced high-frequency asynchronous release of inhibitory transmitter quanta in crayfish nerve-muscle synapses superfused with normal and low-calcium saline

Crayfish fibres of opener muscles were voltage clamped toE=–80 mV membrane potential (T=19–22°C), and veratridine (10–100 mol/l) was added to the superfusate. Within 30–60 s this caused large fluctuations of the clamp current due to vigorous asynchronous quantal release from the inhibitory nerve terminals along the muscle fibre. Excitatory postsynaptic receptors were previously desensitized by application of 5 mmol/l glutamate. Current fluctuations were evaluated by means of the noise analysis technique. Typically, 100 mol/l veratridine increased instantaneously the quantal release rate ñ from ñ<1 quantum/s toñ10,000 quanta/s. Thereafter, ñ declined exponentially with a time constant of 70s. On average, about 500,000 inhibitory quanta could be liberated in this way from the terminals on a single muscle fibre of 1 mm length. Serotonin (1 mol/l) facilitated the effect of lower veratridine concentrations (1–10 mol/l). In opener muscles veratridine-induced asynchronous quantal release showed little dependence on the bath concentration of Ca²⁺. The opposite was found for fibres of the superficial abdominal extensor muscle. Beside postsynaptic current fluctuations, veratridine elicited slowly changing average postsynaptic DC-currents which could be explained partly by superposition of individual inhibitory quantal currents. These DC-currents suggest that beside inhibitory quantal release another factor activates inhibitory postsynaptic receptors after application of veratridine.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Acetylcholine reduces the slow calcium current in embryonic skeletal muscle cells in culture

Xenopus skeletal muscle cells when grown in culture develop a slow inward calcium current that is sensitive to dihydropyridines. Acetylcholine (ACh, 10 M) applied through a puffer pipette caused a large inward current in these cells (at the holding potential) through the nicotinic receptor channels and reduced the inward calcium current (during a step depolarization to 0 mV). After the ACh application was discontinued the holding current rapidly returned to pre-ACh levels (20 s) whereas the calcium current showed a slow, partial recovery to pre-ACh levels. Outward potassium current was also reduced during the application of ACh but recovered completely after ACh was discontinued. The effect of ACh on the calcium current was not mimicked by muscarine (100 M) and was absent when 10 g/ml -bungarotoxin was added to the bath suggesting that the decrease in calcium current was mediated by current through the nicotinic receptor.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.