Hyperosmotic and isosmotic shrinkage differentially affect protein phosphorylation and ion transport

In the present work, we compared the outcome of hyperosmotic and isosmotic shrinkage on ion transport and protein phosphorylation in C11-MDCK cells resembling intercalated cells from collecting ducts and in vascular smooth muscle cells (VSMC) from the rat aorta. Hyperosmotic shrinkage was triggered by cell exposure to hypertonic medium, whereas isosmotic shrinkage was evoked by cell transfer from an hypoosmotic to an isosmotic environment. Despite a similar cell volume decrease of 40%-50%, the consequences of hyperosmotic and isosmotic shrinkage on cellular functions were sharply different. In C11-MDCK and VSMC, hyperosmotic shrinkage completely inhibited Na(+),K(+)-ATPase and Na(+),P(i) cotransport. In contrast, in both types of cells isosmotic shrinkage slightly increased rather than suppressed Na(+),K(+)-ATPase and did not change Na(+),P(i) cotransport. In C11-MDCK cells, phosphorylation of JNK1/2 and Erk1/2 mitogen-activated protein kinases was augmented in hyperosmotically shrunken cells by ～7- and 2-fold, respectively, but was not affected in cells subjected to isosmotic shrinkage. These results demonstrate that the data obtained in cells subjected to hyperosmotic shrinkage cannot be considered as sufficient proof implicating cell volume perturbations in the regulation of cellular functions under isosmotic conditions.     

Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentration

Cytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidant-induced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594-DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure of CGN to a flux of peroxynitrite as low as 0.5-1μM/min during 30min (achieved with 0.1mM SIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca(2+) channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca(2+) dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca(2+) concentration response to the L-VOCC's agonist FPL-64176 and to the L-VOCC's blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca(2+) influx through L-VOCC. Combined analysis of cytosolic Ca(2+) concentration and G-actin/F-actin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.     

Volume changes in cardiac ventricles from Aplysia brasiliana upon exposure to hyposmotic shock

We investigated the possible role of ion channels and transporters in cell volume control using Aplysia brasiliana ventricular tissues exposed to a 26% hyposmotic shock, by assessing changes in wet weight, intracellular water and ionic contents. Thirty minutes after the shock, the wet weight of isolated ventricles increase about 20% above control levels and then attain near original weight within 60 min after the shock. At the time when the wet weight returned to control values, intracellular water and KCl contents are decreased by 22 and 20%, respectively. The K(+) channel blockers, 4-AP and TEA, but not the cotransport blockers, hydrochlorothiazide and furosemide, greatly affect the magnitude of wet weight gain and the time course of weight recovery, indicating that KCl loss occur through conductive pathways. Intracellular recordings performed on ventricular myocytes during exposure to the osmotic shock showed an immediate membrane hyperpolarization and blockade of spontaneous electrical activity; diastolic membrane potential recover over time and spontaneous action potentials are completely restored 60 min after the hyposmotic shock. Because significant weight loss is observed during the exposure of ventricular tissues to 26% hypo-ionic, but isosmotic saline, it is suggested that ventricular volume restoration is accomplished by two distinct but simultaneously occurring processes: a volume-dependent and a volume-independent mechanism. Because wet weight restoration is completely prevented by exposing ventricular tissue to a Ca(2+)-free hyposmotic solution, we postulate that both processes involved in A. brasiliana ventricular weight restoration are Ca(2+)-dependent mechanisms.     

Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes

The effects of short (1 min) and long (7-10 min) exposure to hyposmotic solution on excitation-contraction coupling in rat ventricular myocytes were studied. After short exposure, the action potential duration at 90% repolarization (APD(90)), the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient amplitude, and contraction increased, whereas the L-type Ca(2+) current (I(Ca, L)) amplitude decreased. Fractional sarcoplasmic reticulum (SR) Ca(2+) release increased but SR Ca(2+) load did not. After a long exposure, I(Ca,L), APD(90), [Ca(2+)](i) transient amplitude, and contraction decreased. The abbreviation of APD(90) was partially reversed by 50 microM DIDS, which is consistent with the participation of Cl(-) current activated by swelling. After 10-min exposure to hyposmotic solution in cells labeled with di-8-aminonaphthylethenylpyridinium, t-tubule patterning remained intact, suggesting the loss of de-t-tubulation was not responsible for the fall in I(Ca,L). After long exposure, Ca(2+) load of the SR was not increased, and swelling had no effect on the site-specific phosphorylation of phospholamban, but fractional SR Ca(2+) release was depressed. The initial positive inotropic response to hyposmotic challenge may be accounted for by enhanced coupling between Ca(2+) entry and release. The negative inotropic effect of prolonged exposure can be accounted for by shortening of the action potential duration and a fall in the I(Ca,L) amplitude.     

Osmolyte channel regulation by ionic strength in skate RBC

The aim of this study was to determine whether the opening of the osmolyte channel in skate red blood cells (RBC) is regulated by intracellular electrolyte concentration and conductivity. Consistent with previous studies, experiments with hyperosmotic preincubation before cell swelling or swelling with an isosmotic electrolyte (e.g., ammonium chloride) showed that an increase in ionic strength inhibits the opening of the taurine channel. However, a decrease in intracellular ionic strength did not always stimulate taurine efflux to the same degree. Whereas hyposmotic swelling caused a large increase in taurine efflux, swelling induced by treatment with isosmotic nonelectrolytes produced much smaller stimulation. Results with assays for band 3 phosphorylating enzymes were consistent with those from the taurine efflux studies; stimulation of enzyme activity was lower in cells that were swollen with isosmotic nonelectrolyte media than in cells swollen in hyposmotic media. These results indicate that a decrease in ionic strength is not the only signal for the opening of the taurine channel in skate RBC. Ionic strength does affect channel activity, but there must also be some other regulator.     

Increase of L-type Ca2+ current by protease-activated receptor 2 activation contributes to augmentation of spontaneous uterine contractility in pregnant rats

We evaluated the effects of protease-activated receptor (PAR)-2 on spontaneous myometrial contraction (SMC) in isolated term pregnant myometrial strips of rat, and elucidated the cellular mechanisms of this effect using a conventional voltage-clamp method. In isometric tension measurements, trypsin and SL-NH(2), PAR-2 agonists, significantly augmented SMC in frequency and amplitude; however, boiled trypsin (BT) and LR-NH(2) had no effect on SMC. These stimulatory effects of PAR-2 agonists on SMC were nearly completely occluded by pre-application of Bay K 8644, an L-type voltage-gated Ca(2+) channel activator, thus showing the involvement of L-type voltage-gated Ca(2+) channels in PAR-2-induced augmentation of SMC. In addition, PAR-2 agonists significantly enhanced L-type voltage-gated Ca(2+) currents (I(Ca-L)), as measured by a conventional voltage-clamp method, and this increase was primarily mediated by activation of phospholipase C (PLC) and protein kinase C (PKC) via G-protein activation. Taken together, we have demonstrated that PAR-2 may actively regulate SMC during pregnancy by modulating Ca(2+) influx through L-type voltage-gated Ca(2+) channels, and that this increase of I(Ca-L) may be primarily mediated by PLC and PKC activation. These results suggest a cellular mechanism for the pathophysiological effects of PAR-2 activation on myometrial contractility during pregnancy and provide basic and theoretical information about developing new agents for the treatment of premature labor and other obstetric complications.     

The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles

Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca(2+)](i) of the SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH) induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs. The frequency of the Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular Ca(2+) or in the presence of Ni(2+), the frequency of the Ca(2+) oscillations declined and the airway relaxed. By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching. Each KCl-induced Ca(2+) oscillation consisted of a large Ca(2+) wave that was preceded by multiple localized Ca(2+) transients. KCl-induced responses were resistant to neurotransmitter blockers but were abolished by Ni(2+) or nifedipine and the absence of extracellular Ca(2+). Caffeine abolished the contractile effects of 5-HT, ACH, and KCl. These results indicate that (a) 5-HT and ACH induce airway SMC contraction by initiating Ca(2+) oscillations, (b) KCl induces Ca(2+) transients and twitching by overloading and releasing Ca(2+) from intracellular stores, (c) a sustained, Ni(2+)-sensitive, influx of Ca(2+) mediates the refilling of stores to maintain Ca(2+) oscillations and, in turn, SMC contraction, and (d) the magnitude of sustained airway SMC contraction is regulated by the frequency of Ca(2+) oscillations.     

Biphasic effects of cell volume on excitation-contraction coupling in rabbit ventricular myocytes

We studied the effects of osmotic swelling on the components of excitation-contraction coupling in ventricular myocytes. Myocyte volume rapidly increased 30% in hyposmotic (0.6T) solution and was constant thereafter. Cell shortening transiently increased 31% after 4 min in 0.6T but then decreased to 68% of control after 20 min. In parallel, the L-type Ca(2+) current (I(Ca-L)) transiently increased 10% and then declined to 70% of control. Similar biphasic effects on shortening were observed under current clamp. In contrast, action potential duration was unchanged at 4 min but decreased to 72% of control after 20 min. Ca(2+) transients were measured with fura 2-AM. The emission ratio with excitation at 340 and 380 nm (f(340)/f(380)) decreased by 12% after 3 min in 0.6T, whereas shortening and I(Ca-L) increased at the same time. After 8 min, shortening, I(Ca-L), and the f(340)/f(380) ratio decreased 28, 25, and 59%, respectively. The results suggest that osmotic swelling causes biphasic changes in I(Ca-L) that contribute to its biphasic effects on contraction. In addition, swelling initially appears to reduce the Ca(2+) transient initiated by a given I(Ca-L), and later, both I(Ca-L) and the Ca(2+) transient are inhibited.     

Smooth muscle F-actin disassembly and RhoA/Rho-kinase signaling during endotoxin-induced alterations in pulmonary arterial compliance

Endotoxemia is associated with changed pulmonary vascular function with respect to vasoreactivity, endothelial permeability, and activation of inducible nitric oxide synthase II (NOSII). However, whether altered passive arterial wall mechanics contribute to this endotoxin-induced pulmonary vascular dysfunction is still unknown. Therefore, we investigated whether endotoxin affects the passive arterial mechanics and compliance of isolated rat pulmonary arteries. Pulmonary arteries of pentobarbital-anesthetized Wistar rats (n = 55) were isolated and exposed to Escherichia coli endotoxin (50 microg/ml) for 20 h. Endotoxin increased pulmonary artery diameter and compliance (transmural pressure = 13 mmHg) in an endothelium-, Ca2+-, or NOSII-induced NO release-independent manner. Interestingly, the endotoxin-induced alterations in the passive arterial mechanics were accompanied by disassembly of the smooth muscle cell (SMC) F-actin cytoskeleton. Disassembly of F-actin by incubation of control arteries with the cytoskeleton-disrupting agent cytochalasin B or the Rho-kinase inhibitor Y-27632 induced a similar increase in passive arterial diameter and compliance. In contrast, RhoA activation by lysophosphatidic acid prevented the endotoxin-induced alterations in the pulmonary SMC F-actin cytoskeleton and passive mechanics. In conclusion, these findings indicate that disassembly of the SMC F-actin cytoskeleton and RhoA/Rho-kinase signaling act as mediators of endotoxin-induced changes in the pulmonary arterial mechanics. They imply the involvement of F-actin rearrangement and RhoA/Rho-kinase signaling in endotoxemia-induced vascular lung injury.     

PGE(2) in the regulation of programmed erythrocyte death

Hyperosmotic shock, energy depletion, or removal of extracellular Cl(-) activates Ca(2+)-permeable cation channels in erythrocyte membranes. Subsequent Ca(2+) entry induces erythrocyte shrinkage and exposure of phosphatidylserine (PS) at the erythrocyte surface. PS-exposing cells are engulfed by macrophages. The present study explored the signalling involved. Hyperosmotic shock and Cl(-) removal triggered the release of prostaglandin E(2) (PGE(2)). In whole-cell recording, activation of the cation channels by Cl(-) removal was abolished by the cyclooxygenase inhibitor diclophenac. In FACS analysis, phospholipase-A(2) inhibitors quinacrine and palmitoyltrifluoromethyl-ketone, and cyclooxygenase inhibitors acetylsalicylic acid and diclophenac, blunted the increase of PS exposure following Cl(-) removal. PGE(2) (but not thromboxane) induced cation channel activation, increase in cytosolic Ca(2+) concentration, cell shrinkage, PS exposure, calpain activation, and ankyrin-R degradation. The latter was attenuated by calpain inhibitors-I/II, while PGE(2)-induced PS exposure was not. In conclusion, hyperosmotic shock or Cl(-) removal stimulates erythrocyte PS exposure through PGE(2) formation and subsequent activation of Ca(2+)-permeable cation channels.     

Amino Acids as Osmolytes in the Retina

Amino acids play a role as osmolytes during the regulatory volume decrease subsequent to hyposmotic swelling, but less is known about its role when swelling occurs in isosmotic conditions. In this work we examined the efflux of labelled GABA, taurine and glutamate (traced as D-aspartate) from the chick retina, after isosmotic swelling evoked by KCl-containing solutions, and compared its features to those in hyposmotic swelling. In both conditions, GABA and taurine efflux were more sensitive to swelling than glutamate, as assessed by the activation threshold and the amount released. The amino acid efflux in hyposmotic media was decreased by DIDS, tamoxifen and NPPB, agents acting as Cl channels blockers, which also inhibit the osmosensitive Cl efflux. The component associated with swelling in the KCl-stimulated efflux was assessed by the reduction observed when Cl is replaced by an impermeant anion, or by the influence of hyperosmotic media. GABA and taurine efflux exhibited a large swelling-dependent component, which was lower for D-aspartate. This component was markedly decreased by NPPB, but this was due to an effect of the blocker preventing swelling. These results suggest that the influx of Cl, acting as K counterion, which is responsible for cell swelling, occurs through a pathway sensitive to NPPB, similarly to that activated by hyposmolarity. This finding may be of interest in studies aiming at preventing the cell edema which occurs in a number of pathologies.     

Carbon monoxide protects cardiomyogenic cells against ischemic death through L-type Ca2+ channel inhibition

Carbon monoxide (CO) is known to protect myocardial and vascular cells against injuries due to ischemia-reperfusion or inflammation. We showed that a Ca(2+)-dependent protease calpain promotes necrotic cell death of cardiomyocyte-derived H9c2 cells due to hypoxia through alpha-fodrin proteolysis. Here, we show that ischemia induces necrotic cell death, which is inhibited by either CO, extracellular Ca(2+) deprivation or L-type Ca(2+) channel blockers. A whole cell patch-clamp experiment supports that CO inhibits L-type Ca(2+) channel mediated influx of Ca(2+) and the ischemic death of H9c2 cells.     

Comparisons of different stages of chick embryonic development by the physiological regulatory response to hyposmotic challenge

Cardiac myocytes isolated and cultured from 11 day chick embryos present a Ca(2+)-dependent regulatory volume decrease (RVD) when exposed to hyposmotic stimulus. The RVD of myocytes from different embryonic stages were analyzed to evaluate their physiological performance through development. Among the several embryonic stages analyzed (6, 11, 16 and 19 days) only 19 day cardiac myocytes present a greater RVD when compared with 11 day (considered as control), the other ages showed no difference in the regulatory response. As it is known that RVD is Ca(2+) dependent, we decided to investigate the transient free Ca(2+) response during the hyposmotic swelling of the 11 and 19 day stages. The 11 day cardiac myocyte showed a transient 40% increase in intracellular free Ca(2+) when submitted to hyposmotic solutions, and the free Ca(2+) returned to baseline levels while the cells remained in hyposmotic buffer. However, the intracellular free Ca(2+) transient in the 19 day cells during hyposmotic challenge increases 100% and instead of returning to baseline levels, declines to 55% above control, well after the 11 day transient has returned to baseline. Also, quantitative fluorescence microscopy revealed that 19 day cardiac myocytes have more sarcoplasmic reticulum (SR) Ca(2+) ATPase sites per cell as compared to the 11 day cells. Our findings suggest that 19 day cells have more developed intracellular Ca(2+) stores (SR). By evoking the mechanism of Ca(2+) induced Ca(2+) release, the cells have more free Ca(2+) available for signaling the RVD during hyposmotic swelling.     

Pharmacological modulators of voltage-gated calcium channels and their therapeutical application

Calcium channels (CCs) play an important role in the transduction of action potential to the cytosol. An influx of Ca(2+) is essential for muscle contraction, neurotransmitter, and hormonal release. Level of cytosolic Ca(2+) controls activities of many enzymes and regulatory proteins. Voltage-gated calcium channels (VGCCs) serve as sensors for membrane depolarization. Blood pressure reduction is due to relaxation of actomyosine filaments in vascular smooth muscles. Calcium channel blockers (CCBs) are traditionally used for treatment of cardiovascular diseases. Neurotransmitter release from presynaptic neurons is triggered by Ca(2+) influx. Blockers of neuronal CCs may be applied for pain treatment. Overload of neurons by Ca(2+) is toxic. CCBs may be applied for prevention of some neurodegenerative disorders.     

Spread of discrete structural changes in synthetic polyanionic gel: a model of propagation of a nerve impulse

Thin fibers of cross-linked polyacrylate gel were prepared by inducing polymerization reaction inside long glass or Tygon tubings. By immersing these gel fibers in salt solutions containing both Ca(2+) and Na(+) at varying ratios, a discontinuous transition from the swollen state to the shrunken was demonstrated. A very sharp boundary was observed between the swollen and shrunken portions of the gel fiber. It was found possible to displace this sharp boundary continuously by application of a weak electric current. Based on the similarity in swelling behavior between nerve fibers and synthetic gel fibers, a non-myelinated nerve fiber carrying an impulse was treated as a cylindrical gel layer consisting of two distinct portions, a swollen (active) portion connected directly to the remaining shrunken (resting) portion. By applying the cable theory to this model of the nerve fiber, mathematical expressions describing the conduction velocity, the maximum rate of potential rise, etc. in terms of the electric parameters of the fiber were derived.     

Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder

We have evaluated the presence of capacitative Ca(2+) entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type Ca(2+) channels. Changes in cytosolic Ca(2+) concentration induced by Ca(2+) entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the Ca(2+) store pump, induced a transient Ca(2+) release followed by sustained entry of extracellular Ca(2+). Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the Ca(2+)-mobilizing hormone cholecystokinin octapeptide, or simple removal of external Ca(2+) resulted in a sustained increase in Ca(2+) entry on subsequent reapplication of Ca(2+). This entry was attenuated by 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockade, pinacidil, and Gd(3+). Accumulation of the voltage-sensitive dye 3,3'-dipentylcarbocyanine and direct intracellular recordings showed that depletion of the stores is sufficient for depolarization of the plasma membrane. Contractility studies in intact gallbladder muscle strips showed that CCE induced contractions. The CCE-evoked contraction was sensitive to 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockers, and Gd(3+). We conclude that, in GBSM, release of Ca(2+) from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type Ca(2+) channels. This process may play a role in excitation-contraction coupling in GBSM.     

Targets for calcium channel blockers in mammalian skeletal muscle and their respective functions in excitation-contraction coupling

The L-type Ca2+ channel is blocked by 1,4-dihydropyridines (DHP), by phenylalkylamines, by diphenylbutylpiperidines or by benzolactams. We first show with mouse muscle cells in culture that all these L-type Ca2+ channel blockers block contraction. However, voltage-clamp analysis associated to contraction measurements also clearly show that Ca2+ influx through L-type Ca2+ channels is not required for contraction. Therefore, there is a need for a voltage-sensor which would be responsible for the excitation-contraction (E-C) coupling. We are showing here that the voltage-sensor involved in E-C coupling and the L-type Ca2+ channel have a similar pharmacology. Some of the blockers used are more active on the voltage sensor, others on the L-type Ca2+ channel.     

Na+,K+,2Cl(-)-cotransport and chloride permeability of the cell membrane in mezaton and histamine regulation of electrical and contractile activity in smooth muscle cells from the guinea pig ureter

Influence of Na+,K+,2Cl(-)-cotransport and chloride permeability of the cell membrane on electrically-induced action potential and contraction of smooth muscle cells from guinea pig ureter was examined with the methods of the double sucrose gap junction. Mesatone (10 microM) and histamine (10 microM) induced prolongation of the action potential and elevation of smooth muscle cell contraction, whereas hyperosmic medium (+150 mM sucrose), and recovery of solution osmolality in hyposmic condition (70 mM NaCl) after a single contraction. Inhibitor Na+,K+,2Cl(-)-cotransport bumetanide (10 microM) and chloride permeability blockers niflumic acid (10-100 microM) and SITS (10-500 microM) attenuated stimulating effects of mesatone, histamine and hyperosmic medium. In opposite to adenylate cyclase activation with forskolin (1 microM), guanylate cyclase activation with sodium nitroprusside (SN, 100 microM) decreased both inhibitory action of bumetanide, niflumic acid and activating effects of mesatone, histamine on action potential and elevation contraction of smooth muscle cells. Influence of forskolin rather and not SN on AP and SMC C was inhibited with tetraethylammonium (5 mM). These results suggest that influence of Na+,K+,2Cl(-)-cotransport on electrical and contractil properties of ureter smooth muscle cells is mediated by stimulation of Ca(2+)-activated chloride permeability of the cell membrane and modulated by intracellular cGMP, but not triggered by Ca2+ release from sarcoplasmic reticulum.     

Direct hyposmotic stimulation of gastric acid secretion

Gastric glands isolated from rabbit stomach were incubated in isosmotic medium or media made hyposmotic by 50-100 mOsm/kg. As indicated by radiolabeled aminopyrine accumulation, acid secretion was nearly 3 times greater in 200 mOsm/kg hyposmotic than in isosmotic medium after a 30-min incubation. The hyposmotic stimulation appeared within 2 min, peaked at 10-15 min and declined almost to the isosmotic control by 45 min. As estimated by the wet weight corrected for inulin extracellular space, the intracellular water of the glands also peaked at 15 min and returned to the isosmotic norm by 45 min. Hyposmotic stimulation of acid secretion directly involved the parietal cell, since parietal cells obtained from gastric glands were also stimulated. That the hyposmotic response was direct was indicated by omeprazole inhibition of aminopyrine accumulation in hyposmotic medium.     

Regulation of swelling—activated chloride channels in embryonic chick heart cells

Swelling-activated Cl^- currents,I(Cl,swell),were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique.Genistein,an inhibitor of protein tyrosine kinase(PTK),suppressed I(Cl,swell).Under isosmotic condition phorbol 12-myristate 13-actetate(PMA),and activator of PKC,elicited the Cl^- current similar to that in hyposmotic solution,whereas hyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells,Confocal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition,When the cells were treated with cytochalasin B(CB)or cytochalasin D(CD)under isosmotic condition the disruption of the F-actin integrity was observed,and I(C,l,swell). The results suggested that the role of PTK,probably receptor tyrosine kinase,for regulation of I(Cl,swell) appeared to be at upstream site related to the role of F-actin.Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl^- channels.These results demonstrate clearly that PTK,PKC and F-actin are important factors for regulation of I(Cl,swell),in embryonic chick heart cells as compared with often controversial results reported in different cell types.