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Here we report a 26- to 29-pS cation channel abundantly expressed in freshly isolated and primary cultured type II cells from rat or healthy human lungs. The channel was never spontaneously active in cell-attached patches but could be activated by cell permeabilization with [beta]-escin. Excised patch-clamp experiments revealed activation by [Ca.sup.2+] concentrations at the cytoplasmic side in the micromolar range. High concentrations of amiloride (> 10 [micro]M) at the extracellular side did not inhibit. The channel was equally permeable for K+ and Na+ but was essentially impermeable for [Cl.sup.-] , [Ca.sup.2+], and [Mg.sup.2+]. It was blocked by adenosine nucleotides (cytoplasmic side) with the following order of potency: AMP [aproximately equal to] ADP ([EC.sub.50] [less than or equal to] 10 [micro]M) > ATP [much greater than] adenosine [much greater than] cyclic AMP. The blocking effect of ATP was reproduced by its nonhydrolyzable analogs AMPPNP or ATP-[gamma]-S. GTP did not inhibit. [Cd.sup.2+] blocked the channel with an ECso [approximately or equal to] 55.5 nM. We conclude that type II cells express a [Ca.sup.2+]-dependent, nucleotide-inhibited, nonselective, and [Ca.sup.2+] impermeable cation channel (NS[C.sub.Ca/AMP]) with tonically suppressed activity. RT-PCR confirmed expression of TRPM4b, a channel with functional characteristics almost identical with NS[C.sub.Ca/AMP]. Potential physiological roles are discussed. patch clamp; calcium; adenosine 5'-monophosphate; adenosine 5'diphosphate; adenosine 5'-triphosphate; cadmium; lung; alveolus; nonselective cation channel

Using a new equibiaxial strain device, we investigated strain-induced [Ca.sup.2+] signals and their relation to lamellar body (LB) exocytosis in single rat alveolar type II (AT II) cells. The strain device allows observation of single cells while inducing strain to the entire substratum. AT II cells tolerated high strain amplitudes up to 45% increase in cell surface area ([DELTA]CSA) without release of lactate dehydrogenase or ATP. Strain exceeding a threshold of ~8% [DELTA]CSA resulted in a transient rise of the cytoplasmic [Ca.sup.2+] concentration in some cells. Higher strain levels increased the fraction of [Ca.sup.2+]-responding cells. The occurrence of strain-induced [Ca.sup.2+] signals depended on cell-cell contacts, because lone cells (i.e., cells without cell-cell contacts) did not exhibit [Ca.sup.2+] signals. Above threshold, the amplitude of the [Ca.sup.2+] signal as well as the number of stimulated LB fusions correlated well with the amplitude of strain. Furthermore, stimulated LB fusions occurred only in cells exhibiting a [Ca.sup.2+] signal; 50 [micro]M [Gd.sup.3+] in the bath affected neither [Ca.sup.2+] signals nor fusions. Intracellular [Ca.sup.2+] release was triggered at higher strain amplitudes and inhibited by thapsigargin. Removal of bath [Ca.sup.2+] completely inhibited [Ca.sup.2+] signals and fusions. We conclude that strain of AT II cells stimulates a [Ca.sup.2+] entry pathway that is highly sensitive to strain and a prerequisite for subsequent [Ca.sup.2+] release. Both mechanisms result in a graded response of fusions to strain. Our data also allow us to introduce the term 'effective strain' as the physiologically relevant portion of the strain amplitude. surfactant secretion; alveolar type II cells; mechanical strain; stretch

The feasibility of using the dye FM 1-43 to measure lamellar body exocytosis at high spatial and temporal resolution in alveolar type II cells was investigated. Fluorescence microscopy was combined with the whole cell patch-clamp approach to determine how the FM 1-43 fluorescence gain reflects the instance and number of fusion events or which factors affect the time course of the FM 1-43 gain. Experimental results showed that the FM 1-43 technique is highly suitable for measurement of the onset of exocytosis and amount of secretion in alveolar type II cells.

Abstract Oncostatin M (OSM) is a member of the interleukin-6 cytokine family and regulates eg. gene activation, cell survival, proliferation and differentiation. OSM binds to a receptor complex consisting of the ubiquitously expressed signal transducer gp130 and the ligand binding OSM receptor subunit, which is expressed on a specific subset of primary afferent neurons. In the present study, the effect of OSM on heat nociception was investigated in nociceptor-specific gp130 knock-out (SNS-gp130-/-) and gp130 floxed (gp130fl/fl) mice. Subcutaneous injection of pathophysiologically relevant concentrations of OSM into the hind-paw of C57BL6J wild type mice significantly reduced paw withdrawal latencies to heat stimulation. In contrast to gp130fl/fl mice, OSM did not induce heat hypersensitivity in vivo in SNS-gp130-/- mice. OSM applied at the receptive fields of sensory neurons in in vitro skin-nerve preparations showed that OSM significantly increased the discharge rate during a standard ramp-shaped heat stimulus. The capsaicin- and heat-sensitive ion channel TRPV1, expressed on a subpopulation of nociceptive neurons, has been shown to play an important role in inflammation-induced heat hypersensitivity. Stimulation of cultured dorsal root ganglion neurons with OSM resulted in potentiation of capsaicin induced ionic currents. In line with these recordings, mice with a null mutation of the TRPV1 gene did not show any signs of OSM-induced heat hypersensitivity in vivo. The present data suggest that OSM induces thermal hypersensitivity by directly sensitizing nociceptors via OSMR-gp130 receptor mediated potentiation of TRPV1.

Haller, Thomas, Klaus Auktor, Manfred Frick, Norbert Mair, and Paul Dietl. Threshold calcium levels for lamellar body exocytosis in type II pneumocytes. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L893-L900, 1999.--Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]].sub.i]). In fura 2-loaded cells, [[Ca.sup.2+]].sub.i] was selectively elevated by flash photolysis of a cell-permeant caged [Ca.sup.2+] compound (o-nitrophenyl EGTA-AM) or by gradually enhancing cellular [Ca.sup.2+] influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579-1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [[Ca.sup.2+]].sub.i] elevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [[Ca.sup.2+]].sub.i] and thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize [Ca.sup.2+]. This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced [Ca.sup.2+] signal below ~300 nmol/l. Our results suggest a highly [Ca.sup.2+]-sensitive step in LB exocytosis. surfactant secretion; alveolar type II cells; flash photolysis; caged calcium

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579-1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic [Ca.sup.2+] concentration. Consistently, content release correlated with the occurrence of [Ca.sup.2+] oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca2+ concentration ([Ca2+])c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca2+ entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X4 receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X4. Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca2+ influx via vesicular Ca2+ channels regulates fusion pore expansion and vesicle content release. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P1 receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P1 receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P1 receptor. Our data show that neuronally expressed S1P1 receptors play a significant role in regulating nociceptor function and that S1P/S1P1 signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation. [ABSTRACT FROM AUTHOR]

To provide a tool to investigate the mechanisms inducing and maintaining cancer-related pain and hyperalgesia, a soft tissue tumor/ metastasis model was developed that is applicable in C57BL/6J wild-type and transgenic mice. We show that the experimental tumor/induced heat hyperalgesia and nociceptor sensitization were prevented by systemic treatment with the tumor necrosis factor α (TNFα) antagonist etanercept. In naive mice, exogenous TNF α evoked heat hyperalgesia in vivo and sensitized nociceptive nerve fibers to heat in vitro. TNFα enhanced the expression of the nociceptor-specific heat transducer ion channel transient receptor potential vanilloid 1 (TRPV1) and increased the amplitudes of capsaicin and heat-activated ionic currents via p38/MAP (mitogen -activated protein) kinase and PKC (protein kinase C). Deletion of the tumor necrosis factor receptor type 2 (TNFR2) gene attenuated heat hyperalgesia and prevented TRPV1 upregulation in tumor-bearing mice, whereas TNFR1 gene deletion played a minor role. We propose endogenous TNFα as a key player in cancer-related heat hyperalgesia and nociceptor sensitization that generates TRPV1 upregulation and sensitization via TNFR2. [ABSTRACT FROM AUTHOR]

Presents a study that used an equibiaxial strain device to investigate strain-induced calcium ion Ca2+ signals and their relation to lamellar body exocytosis in single rat alveolar type II cells. Toleration of high strain amplitudes up to 45 percent increase in cell surface area without release of lactase dehydrogenase; Transient rise of cytoplasmic calcium ion concentration in some cells.

Presents information on a study which examined the capabilities of lamellar body exocytosis by measurement of cell membrane capacitance and laser-scanning confocal microscopy. Methodology; Results and discussion.

Background: Hemoglobin‑oxygen (Hb-O 2 ) affinity is an important determinant for oxygen delivery and oxygen extraction. Although cardiovascular agents such as noradrenaline, adrenaline, atropine, milrinone and levosimendan are widely used in intensive care units worldwide, nothing is known about their possible effects on Hb-O 2 affinity., Methods: In this experimental ex-vivo trial, venous blood samples were taken from 5 male and 6 female volunteers and incubated with the particular cardiovascular agents. Oxygen dissociation curves (ODC) were measured in-vitro with a new high-throughput method., Results: Compared to the P 50 in male and female controls, a significant right-shift of the ODC was found for noradrenaline and milrinone in all participants and for levosimendan in male samples only. Adrenaline decreased Hb-O 2 affinity in male samples, atropine in female samples only., Discussion: All investigated agents decreased Hb-O 2 affinity, with marked differences between males and females. Although the underlying mechanisms remain unclear, the extent of these effects may increase oxygen extraction at the tissue level as long as pulmonary oxygen uptake is maintained., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Inhalational prostacyclins act as strong vasodilators, potentially improving oxygenation by reducing shunt fraction and ventilation-perfusion mismatch. As prostacyclin receptors are known to be present on human erythrocytes, possible direct effects on hemoglobin oxygen transport were further explored by examining the sole in vitro influence of prostacyclins on hemoglobin oxygen (Hb-O 2 ) affinity. Venous blood samples from 20 healthy volunteers were exposed in vitro to supramaximal doses of epoprostenol, iloprost, and compared with control. By high-throughput measurements, hemoglobin oxygen dissociation curves (ODCs) were derived. Hb-O 2 affinity, expressed by P50 and Hill coefficient, was determined and analyzed for three subgroups: males ( n = 10), females not taking oral contraceptives ( n = 4), and females taking oral contraceptives ( n = 6). Epoprostenol significantly decreased P50 in all (males, females without contraceptives, and females taking oral contraceptives) [27.5 (26.4-28.6) mmHg (control) vs. 24.2 (22.7-25.3) mmHg; P < 0.001. median (interquartile range, IQR)] thereby increasing Hb-O 2 affinity. Inversely, iloprost only showed significant effects in females taking oral contraceptives where P50 was markedly increased and therefore Hb-O 2 affinity decreased [28.4 (27.9-28.9) mmHg (control) vs. 34.4 (32.2-36.0) mmHg; P < 0.001]. Prostacyclin-receptor stimulation and subsequent cAMP-mediated ATP release from erythrocytes are discussed as a possible underlying mechanism for the effect of epoprostenol on Hb-O 2 affinity. The reason for the sex hormone-modified iloprost effect remains unclear. Being aware of potentially differing effects on Hb-O 2 affinity might help select the right prostacyclin (epoprostenol vs. iloprost) depending on the patient and the underlying disease (e.g., acute respiratory distress syndrome vs. peripheral arterial disease).

Ca(2+) is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca(2+) concentration ([Ca(2+)])(c) leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca(2+) channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca(2+) entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca(2+) entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca(2+) channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X(4) receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X(4) receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X(4). Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca(2+) influx via vesicular Ca(2+) channels regulates fusion pore expansion and vesicle content release.