Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland.

The CLCA family of Ca2+-activated Cl- channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.

Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated Cl- channel proteins.

We have cloned and molecularly and functionally characterized the first human member of the family of Ca2+-activated Cl- channels, human (h) CLCA1. The 31,902-bp gene is located on chromosome 1p22-31 and is preceded by a canonic promoter region that contains an L1 transposable element. In contrast to all previously known homologs in other species, hCLCA1 is exclusively expressed in intestinal basal crypt epithelia and goblet cells, suggesting that it does not represent the human counterpart of any of them. Expression of the 914-amino-acid hCLCA1 protein in HEK 293 cells yielded a 125-kDa precursor that was processed to yield two cell-surface-associated subunits, a 90-kDa protein and a group of 37- to 41-kDa proteins. Four transmembrane domains were established within the 90-kDa subunit. HEK 293 cells transfected with CLCA1 exhibited an increase in whole-cell Ca2+-sensitive Cl- currents that were outwardly rectified and inhibited by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid, dithiothreitol, and niflumic acid. Cell-attached patch recordings of transfected cells revealed single channels with a slope conductance of 13.4 pS. These findings suggest that human CLCA1 mediates a Ca2+-activated Cl- conductance in the human intestine and make it an interesting candidate as a modulating factor in the pathogenesis of cystic fibrosis.

Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung.

A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.

Involvement of ceramide in inhibitory effect of IL-1 beta on L-type Ca2+ current in adult rat ventricular myocytes.

Interleukin (IL)-1 beta has previously been shown to decrease the L-type Ca2+ channel current (ICa,L). Because ceramide has been suggested to mediate many biological effects of IL-1 beta, we examined whether ceramide was involved in the IL-1 beta-induced suppression of ICa,L in adult rat ventricular myocytes. Exposure of myocytes to 5 ng/ml IL-1 beta elicited a 140% increase in ceramide production within 2 min, as measured with 32P phosphorylation. Whole cell patch-clamp techniques were used to measure ICa,L in myocytes internally dialyzed and externally perfused with Na(+)- and K(+)-free solutions. C2 ceramide (1 nM-1 microM), a membrane-permeable analog of ceramide, caused a concentration-dependent inhibition of ICa,L and increased the rate of ICa,L inactivation without altering its gating properties. An inactive ceramide analog failed to inhibit ICa,L. At submaximal concentrations, effects of C2 ceramide and IL-1 beta on ICa,L were additive and saturable. In the presence of a maximally effective concentration of IL-1 beta, C2 ceramide had no further effect on ICa,L. These results suggest that ceramide mediates IL-1 beta-induced suppression of cardiac ICa,L.

1,2-Dioctanoyl-sn-glycerol depresses cardiac L-type Ca2+ current: independent of protein kinase C activation.

The present study examines the effect of 1,2-dioctanoyl-sn-glycerol (DiC8), a diacylglycerol analogue, on L-type Ca2+ current (ICa,L) in adult rat ventricular myocytes using whole cell patch-clamp techniques. Extracellular application of DiC8 (1-10 microM) resulted in a concentration-dependent inhibition of peak ICa,L (half-maximum inhibitory concentration = 2.2 microM). Results obtained from the current-voltage relationship showed that DiC8 decreased the slope conductance. In addition, DiC8 increased the rate of Ba2+ current inactivation and caused a hyperpolarizing shift in the steady-state inactivation by 6 mV and a decrease in the slope factor. The DiC8-induced inhibition of ICa,L was neither mimicked by activation of protein kinase C (PKC) with 100 nM phorbol 12-myristate 13-acetate (PMA) no prevented by inhibition of PKC with 30 microM H-7, 100 nM staurosporine, or 24-h pretreatment with PMA. These results suggest that in rat ventricular myocytes 1) 1,2-sn-diacylglycerol (DAG) inhibits ICa,L, possibly by facilitating channel inactivation and decreasing channel availability and 2) this inhibitory effect of DAG is independent of PKC activation.

Acetylcholine induces contraction in vergebral arteries from treated hypertensive patients.

Endothelium-dependent vasodilatation to acetylcholine is abnormal in animal models of hypertension. This abnormality reflects a change in the balance of relaxing and contracting factors produced in the vascular wall. In human cerebral arteries, endothelin has been implicated in the abnormal vasoconstrictor response following subarachnoid hemorrhage. This study tests the hypothesis that cerebral arteriolar dilatation to acetylcholine reduced in clinical hypertension due to an overproduction of endothelin. Our results show that at high concentrations of muscarinic agonist (0.3-3 microM), human vertebral arteries from hypertensive patients contract whereas those from normotensive patients remain maximally dilated. We conclude that the normal dilator response to acetylcholine is abrogated in vertebral arteries from treated hypertensive patients but endothelin-1 does not contribute to the abnormal responsiveness.

G protein-mediated suppression of L-type Ca2+ current by interleukin-1 beta in cultured rat ventricular myocytes.

The effect and possible signal transduction pathway of interleukin-1 beta (IL-1 beta) on the L-type Ca2+ current (ICa,L) in cultured adult rat ventricular myocytes were examined using whole cell patch-clamp techniques. When myocytes were internally dialyzed with a solution containing GTP, IL-1 beta caused a concentration-dependent decrease in the peak ICa,L (Ba2+ as the charge carrier). IL-1 beta did not significantly alter the voltage dependence of the peak ICa,L nor the steady-state inactivation and activation, but did slightly slow the rate of inactivation. In myocytes dialyzed with solutions without GTP or including guanosine 5'-O-(2-thiodiphosphate) to replace GTP, IL-1 beta had no effect on ICa,L. In contrast, when guanosine 5'-O-(3-thiotriphosphate) was used to replace GTP, the suppression of ICa,L induced by IL-1 beta remained. Preincubation of myocytes with pertussis toxin (PTX), which completely abolished the acetylcholine effect on isoproterenol-stimulated ICa,L, had no effect on the inhibitory action of IL-1 beta on ICa,L. We conclude that in cultured rat ventricular myocytes, IL-1 beta suppresses ICa,L via a PTX-insensitive G protein.

Endothelium dependency of contractile activity differs in infant and adult vertebral arteries.

Contractions to serotonin (5-HT) and endothelin-1 (ET-1) in infant (0-2 yr) and adult (38-71 yr) vertebral arteries were examined in the presence of either the cyclooxygenase inhibitor indomethacin or NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide production. In addition, endothelium-dependent relaxations to acetylcholine were characterized in arteries contracted with agonist. The results showed that: (a) Contractions of infant arteries to 5-HT or ET-1 decreased to 44 +/- 8% and 27 +/- 13%, respectively, within 10 min. Indomethacin or removal of endothelium abolished this decreased response, whereas L-NMMA had no effect. (b) Adult arteries produced sustained contractions to 5-HT or ET-1 that were unaffected by indomethacin, endothelium denudation, or L-NMMA. (c) Endothelium-dependent relaxations to acetylcholine were greater in infant than adult arteries and were abolished by indomethacin (but not L-NMMA) in infants and L-NMMA (but not indomethacin) in adults. Thus, endothelium-dependent responses in infant arteries are attenuated because of increased prostaglandin activity not observed in adult tissues. Additionally, there is an age-dependent change in the primary mechanism responsible for acetylcholine-induced vasodilation. Apparently, endothelium dependency of acetylcholine-induced relaxation is highly dependent on cyclooxygenase activity in the infant vertebral artery, but in the adult artery, nitric oxide is linked to the vasodilator response.

Oscillatory contractions in vertebral arteries from hypertensive subjects.

In response to several agonists, tail arteries from spontaneously hypertensive stroke prone rats (SHRSP) contract in an oscillatory manner not observed in tail arteries from normotensive rats. This study evaluated whether oscillations in force development characterize the contractile pattern of vertebral arteries from hypertensive humans. Vertebral arteries were isolated and studied within 18-24 h post mortem. Helical strips of the arteries were mounted in a muscle bath for isometric force recording. Contractile responses to serotonin (10(-7)M) and endothelin (10(-8)M) in arteries from hypertensive subjects were characterized by fluctuations in force development whereas those in arteries from normotensive subjects usually remained constant with time. The frequency of the response was approximately 1-2 contraction/relaxation cycles per min. This pattern of oscillatory contractile activity was observed in all but one of the hypertensive arteries (n = 15), and in approximately 40% of the normotensive arteries (n = 12). Oscillatory activity was converted to maintained contraction by nifedipine (10(-7)M) which also caused relaxation of the contractile response. Relaxation to acetylcholine (10(-6)M) and nitroglycerin (10(-6)M) did not alter the oscillatory contractions. Endothelium removal did not influence the oscillatory pattern of contraction. These observations suggest that oscillatory contractile activity in vertebral arteries from hypertensive subjects is related to abnormal calcium entry into the smooth muscle cells. This altered membrane property may contribute to changes in vascular reactivity in hypertension.