Zvi Priel

PURINERGIC STIMULATION OF RABBIT CILIATED AIRWAY EPITHELIA : CONTROL BY MULTIPLE CALCIUM SOURCES

1. Simultaneous measurements of average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were carried out on ciliated rabbit tracheal cells in order to determine quantitatively the role of calcium in the regulation of mucus‐transporting cilia. 2. Extracellular ATP caused a rapid increase in both [Ca2+]i and CBF in the 0.1‐1000 microM concentration range. The rise in [Ca2+]i levelled off to an elevated [Ca2+]i plateau while the cilia remained in a high activation state. The magnitude of the rise in [Ca2+]i and CBF as well as the value of the elevated [Ca2+]i plateau and the value of the sustained CBF were dependent on the concentration of ATP in the solution. 3. No correlation was found between the mean values of [Ca2+]i and CBF at rest but a sigmoidal relationship was found to exist between the maximal rises of these parameters following excitation with extracellular ATP. This sigmoidal correlation incorporated the experiments where [Ca2+]i rise was induced by depletion of internal calcium stores with thapsigargin or by entry of calcium induced by ionomycin. 4. Extracellular ATP caused both the release of calcium from internal stores and calcium influx from the extracellular solution. The release of calcium was identified as originating from a thapsigargin‐sensitive and a thapsigargin‐insensitive calcium store. It is suggested that the release of calcium from these stores induces the initial rise in CBF. 5. The sustained activation of the cilia and elevated calcium plateau were found to be the result of the extracellular ATP‐induced calcium influx. This calcium influx was insensitive to the voltage‐gated calcium channel inhibitors verapamil and diltiazem, but was completely eliminated by lowering the extracellular calcium concentration to 0.1 microM. 6. We propose that the initial jump in the CBF is mediated by the calcium released from a thapsigargin‐insensitive calcium store adjacent to the cilia, while the later, and longer, rise in CBF is the result of the calcium emanating from the thapsigargin‐sensitive store which is positioned further away from the cilia within the cell cytoplasm. The calcium influx that follows is responsible for sustaining the cilia at a high level of excitation.

Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea

1 Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation. 2 Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U‐73122) or by suramin. 3 The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP‐adenosine 3′, 5′‐cyclic monophosphothioate triethylamine (Rp‐cAMPS). 4 Activation of PKC by phorbol 12‐myristate, 13‐acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl‐cAMP led to a small rise in CBF without affecting [Ca2+]i. 5 Direct activation of protein kinase G (PKG) with dibutyryl‐cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl‐cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin. 6 The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP.

Extracellular ATP directly gates a cation-selective channel in rabbit airway ciliated epithelial cells

1 A membrane conductance activated by extracellular ATP was identified and characterized in freshly dissociated rabbit airway ciliated cells using the whole‐cell and outside‐out patch configurations of the patch‐clamp technique. 2 In solutions designed to maximize currents through voltage‐gated calcium channels, there were no indications of voltage‐gated Ba2+ currents. 3 Extracellular ATP (but not UTP or ADP) activated a membrane conductance which remained activated for several minutes in the presence of ATP. The conductance was permeable to monovalent and divalent cations with approximate relative permeabilities (P) for PBa:PCs:PTEA of 4:1:0.1. Permeability to Cl− was negligible. 4 Including GDP‐β‐S in the intracellular solution did not inhibit the effects of ATP, nor did GTP‐γ‐S irreversibly activate the conductance. 5 In outside‐out membrane patches, with GDP‐β‐S in the pipette solution, ATP activated ion channels which had a chord conductance of approximately 6 pS in symmetrical 150 mM CsCl solutions at ‐120 mV. 6 Suramin (100 μM) inhibited the whole‐cell currents activated by ATP (200 μM) by 93 ± 3 %. Similar effects of suramin were observed on ATP‐activated channels in outside‐out membrane patches. 7 Extracellular ATP had a priming action on the response to subsequent exposure to ATP. At ‐40 mV, the time to half‐maximal current activation (t½) was 46 ± 9 s during the first exposure to 200 μM ATP and decreased to 5 ± 3 s during a second exposure to the same concentration of ATP. The priming action of ATP was not inhibited by including GDP‐β‐S in the intracellular solution. 8 The initial rate of activation increased with the concentration of ATP, and was voltage sensitive. During the first exposure to 200 μM ATP, t½ at +40 mV was 4‐fold longer than t½ at ‐40 mV. 9 Half‐maximal activation of the conductance shifted from 210 ± 30 to 14 ± 4 μM added ATP when CaCl2 in the extracellular solution was reduced from 1.58 to 0.01 mM. The Hill coefficient for ATP was 1.2 in both solutions. 10 These observations suggest that a form of ATP uncomplexed with divalent cations directly gates an ion channel (P2X receptor) in rabbit airway ciliated cells, which serves as a pathway for Ca2+ influx. This purinoceptor may contribute to sustained ciliary activation during prolonged exposures to ATP.

Extracellular sodium regulates airway ciliary motility by inhibiting a P2X receptor

The mucociliary system is responsible for clearing inhaled particles and pathogens from the airways. This important task is performed by the beating of cilia and the consequent movement of mucus from the lungs to the upper airways,. Because ciliary motility is enhanced by elevated intracellular calcium concentrations, inhibition of calcium influx could lead to disease by jeopardizing mucociliary clearance. Several hormones and neurotransmitters stimulate ciliary motility, one of the most potent of which is extracellular ATP (ATPo), which acts by releasing calcium ions from internal stores and by activating calcium influx. Here we show that, in airway ciliated cells, extracellular sodium ions (Nao+) specifically and competitively inhibit an ATPo-gated channel that is permeable to calcium ions, and thereby attenuate ATPo-induced ciliary motility. Our finding points to a physiological role for Nao+ in ciliary function, and indicates that mucociliary clearance might be improved in respiratory disorders such as chronic bronchitis and cystic fibrosis by decreasing the sodium concentration of the airway surface fluid in which the cilia are bathed.

Protein kinase C induced calcium influx and sustained enhancement of ciliary beating by extracellular ATP

The major purpose of this work was to determine protein kinase C (PKC) influence on ciliary beat frequency (CBF) and to assess participation of PKC in purinergic ciliary stimulation. The experiments were performed by simultaneous measurement of [Ca2+]i and CBF on tissue culture of frog esophagus epithelium. The PKC activators TPA and DiC8 produced significant elevation of [Ca2+]i and strong frequency enhancement. The calcium elevation was inhibited by lowering the extracellular calcium level, or by La3+, but was unaffected by verapamil and the phospholipase C inhibitor U-73122, suggesting that Ca2+ influx was via non-voltage-operated calcium channels. The inhibition of [Ca2+]i elevation resulted in corresponding inhibition of CBF enhancement. The effect of TPA was blocked by the selective PKC inhibitors chelerythrine, calphostin C, and GF109203X, and by the enzyme downregulation. The downregulation of PKC, or the enzyme inhibitors did not affect the immediate response to extracellular ATP but caused rapid decay of initially stimulated [Ca2+]i and CBF to the basal level. These results suggest that PKC produces CBF enhancement via activation of calcium influx through non-voltage-operated calcium channels. This calcium influx seems to be responsible for the duration of ciliary stimulation produced by the extracellular ATP.

Simultaneous measurement of ciliary beating and intracellular calcium

A novel system for measuring, simultaneously, ciliary beating and intracellular free calcium is presented. The advantages and dynamic nature of the system are demonstrated by measuring the effects of the calcium ionophore lonomycin and of extracellular ATP on ciliated rabbit trachea. The results are discussed with regard to the ciliary and calcium stimulation.

The Mechanism of Ciliary Stimulation by Acetylcholine: Roles of Calcium, PKA, and PKG

Stimulation of ciliary cells through muscarinic receptors leads to a strong biphasic enhancement of ciliary beat frequency (CBF). The main goal of this work is to delineate the chain of molecular events that lead to the enhancement of CBF induced by acetylcholine (ACh). Here we show that the Ca2+, cGMP, and cAMP signaling pathways are intimately interconnected in the process of cholinergic ciliary stimulation. ACh induces profound time-dependent increase in cGMP and cAMP concentrations mediated by the calcium–calmodulin complex. The initial strong CBF enhancement in response to ACh is mainly governed by PKG and elevated calcium. The second phase of CBF enhancement induced by ACh, a stable moderately elevated CBF, is mainly regulated by PKA in a Ca2+-independent manner. Inhibition of either guanylate cyclase or of PKG partially attenuates the response to ACh of [Ca2+]i, but completely abolishes the response of CBF. Inhibition of PKA moderately attenuates and significantly shortens the responses to ACh of both [Ca2+]i and CBF. In addition, PKA facilitates the elevation in [Ca2+]i and cGMP levels induced by ACh, whereas an unimpeded PKG activity is essential for CBF enhancement mediated by either Ca2+ or PKA.

Irreversible adsorption of triple-helical soluble collagen monomers from solution to glass and other surfaces

The adsorption from various solutions of triple-helical soluble collagen monomers to solid surfaces was studied by labeling the collagen with 1251. Adsorption to glass, siliconized glass, and Teflon, from aqueous solutions at various pH and ionic strength, was determined at collagen concentrations from 2 to 25 μg/ml. Adsorption was shown to be irreversible and little dependent on pH and ionic strength but increasing enormously as the surface is made more hydrophobic. Surface denaturation of the collagen by heat results in a substantial loss of material. The kinetics of adsorption suggest that the adsorption process may be selective and that not all collagen molecules which reach the surface are immediately adsorbed. Checking these results with earlier measurements of adsorbed layer thickness, a model for collagen adsorption is proposed.

Intracellular Ca2+ Regulates the Phosphorylation and the Dephosphorylation of Ciliary Proteins Via the NO Pathway

The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO syntase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating.

Possible mechanism of ciliary stimulation by extracellular ATP: involvement of calcium-dependent potassium channels and exogenous Ca2+.

SummaryCiliary motility was examined optically in tissue cultures from frog palate epithelium and frogs esophagus as a function of extracellular concentration of adenosine 5′-triphosphate (ATP) and related compounds. The addition of micromolar concentration of ATP caused a strong enhancement of frequency and wave velocity in the direction of the effective stroke. Since adenosine 5′-[β,γ imido]-triphosphate (AMP-PNP), a nonhydrolyzable analog of ATP, produces the same effects, ATP hydrolysis is not required. The overall potency is ATP ≅ AMP-PNP>ADP ≫adenosine>AMP. It is suggested that both the phosphate and the base moieties are involved in ATP binding.The enhancement of ciliary activity by extracellular ATP is dependent on the presence of extracellular Ca2+, which can be replaced by extracellular Mg2+. The effect of a number of potent inhibitors of the voltage-gated calcium channels on the stimulation of ciliary activity by ATP were examined. No effect was detected in the concentration range within which these agents are specific. On the other hand, quinidine, a potent inhibitor of K+ (calcium-dependent) channels, inhibits the effect of ATP.The following model is suggested: exogenous ATP interacts with a membrane receptor in the presence of Ca2+, a cascade of events occurs which mobilizes intracellular calcium, thereby increasing the cytosolic free Ca2+ concentration which consequently opens the calcium-activated K+ channels, which then leads to a change in membrane potential. The ciliary response to these changes is the enhancement of ciliary activity.