Influence of some phospholipase A2 and cytochrome P450 inhibitors on rat arterial smooth muscle K+ currents.

The hyperpolarizing factor that is liberated by vascular endothelial cells in response to various agonists, and known to induce relaxation by opening of smooth muscle K+ channels, has been suggested to be a product of cytochrome P450 dependent arachidonic acid metabolism. In this study, the direct influence of two phospholipase A2 inhibitors and of five structurally and mechanistically different cytochrome P450 inhibitors on K+ currents in freshly isolated vascular smooth muscle cells from the rat aorta was investigated. On stepping the cell membrane potential from -70 mV to a series of depolarized test potentials, a noisy outward current developed at test potentials > +10 mV, which showed no appreciable inactivation during the voltage pulse. It was largely abolished by 3 mM external tetraethylammonium chloride (TEA), suggesting that it predominantly consisted of current through large-conductance Ca(2+)-activated K+ channels. The phospholipase A2 inhibitor quinacrine considerably inhibited this TEA-sensitive current, while 4-bromophenacylbromide exerted no effect. The cytochrome P450 inhibitors proadifen and miconazole reversibly decreased the amplitude of I(K), while clotrimazole and 1-aminobenzotriazole had no effect. Conversely, 17-octadecynoic acid increased whole-cell I(K). These results show that some phospholipase A2 and cytochrome P450 inhibitors may interfere with K+ channel activation in the rat arterial smooth muscle cell. The relevance of these findings to studies on the involvement of cytochrome P450 dependent metabolism in the generation of the endothelium-derived hyperpolarizing factor in intact arteries is discussed.     

Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide-gated ion channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory transduction in olfactory sensory neurons and retinal photoreceptor cells. In these systems, internal calcium/calmodulin (Ca2+/CaM) inhibits CNG channels, thereby having a putative role in sensory adaptation. Functional differences in Ca2+/CaM-dependent inhibition depend on the different subunit composition of olfactory and rod CNG channels. Recent evidence shows that three subunit types (CNGA2, CNGA4, and CNGB1b) make up native olfactory CNG channels and account for the fast inhibition of native channels by Ca2+/CaM. In contrast, two subunit types (CNGA1 and CNGB1) appear sufficient to mirror the native properties of rod CNG channels, including the inhibition by Ca2+/CaM. Within CNG channel tetramers, specific subunit interactions also mediate Ca2+/CaM-dependent inhibition. In olfactory CNGA2 channels, Ca2+/CaM binds to an N-terminal region and disrupts an interaction between the N- and C-terminal regions, causing inhibition. Ca2+/CaM also binds the N-terminal region of CNGB1 subunits and disrupts an intersubunit, N- and C-terminal interaction between CNGB1 and CNGA1 subunits in rod channels. However, the precise N- and C-terminal regions that form these interactions in olfactory channels are different from those in rod channels. Here, we will review recent advances in understanding the subunit composition and the mechanisms and roles for Ca2+/CaM-dependent inhibition in olfactory and rod CNG channels.     

Cloning of a gap junctional protein from vascular smooth muscle and expression in two-cell mouse embryos.

Gap junctional proteins (connexins) form aqueous channels that enable direct cell-cell transfer of ions and small molecules. The distribution and conductance of gap junction channels in cardiac muscle determine the pattern and synchrony of cellular activation. However, the capacity for smooth muscle to restrict contractile events temporally and spatially suggests that cell-cell coupling or its regulation may be decidedly different in this tissue. We isolated a cDNA from vascular smooth muscle which encodes a connexin (Mr 43,187) structurally homologous to cardiac connexin43. Vascular smooth muscle connexin43 mRNA was expressed prominently in smooth muscle tissues, cultured vascular myocytes, and arterial endothelial cells. A model for functional expression of connexins was developed in two-cell B6D2 mouse embryos. Microinjection of in vitro transcribed vascular smooth muscle connexin43 mRNA was shown to be sufficient to induce intercellular coupling in previously uncoupled blastomeres. Through the construction of two deletion mutants of connexin43, we also show that the formation of cell-to-cell connections does not depend upon a predicted cytoplasmic region within 98 residues of the carboxyl terminus. Finally, the identification of connexin43 in smooth muscle and endothelial cells provides supporting evidence for the existence of heterocellular coupling between cells of the vascular intima.     

Functionally important calmodulin-binding sites in both NH2- and COOH-terminal regions of the cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit

Whereas an important aspect of sensory adaptation in rod photoreceptors and olfactory receptor neurons is thought to be the regulation of cyclic nucleotide-gated (CNG) channel activity by calcium-calmodulin (Ca2+-CaM), it is not clear that cone photoreceptor CNG channels are similarly modulated. Cone CNG channels are composed of at least two different subunit types, CNGA3 and CNGB3. We have investigated whether calmodulin modulates the activity of these channels by direct binding to the CNGB3 subunit. Heteromeric channels were formed by co-expression of human CNGB3 with human CNGA3 subunits in Xenopus oocytes; CNGB3 subunits conferred sensitivity to regulation by Ca2+-CaM, whereas CaM regulation of homomeric CNGA3 channels was not detected. To explore the mechanism underlying this regulation, we localized potential CaM-binding sites in both NH2- and COOH-terminal cytoplasmic domains of CNGB3 using gel-overlay and glutathione S-transferase pull-down assays. For both sites, binding of CaM depended on the presence of Ca2+. Individual deletions of either CaM-binding site in CNGB3 generated channels that remained sensitive to regulation by Ca2+-CaM, but deletion of both together resulted in heteromeric channels that were not modulated. Thus, both NH2- and COOH-terminal CaM-binding sites in CNGB3 are functionally important for regulation of recombinant cone CNG channels. These studies suggest a potential role for direct binding and unbinding of Ca2+-CaM to human CNGB3 during cone photoreceptor adaptation and recovery.     

Vanilloid Receptor-1 (TRPV1) Expression and Function in the Vasculature of the Rat

Transient receptor potential (TRP) cation channels are emerging in vascular biology. In particular, the expression of the capsaicin receptor (TRPV1) was reported in vascular smooth muscle cells. This study characterized the arteriolar TRPV1 function and expression in the rat. TRPV1 mRNA was expressed in various vascular beds. Six commercially available antibodies were tested for TRPV1 specificity. Two of them were specific (immunostaining was abolished by blocking peptides) for neuronal TRPV1 and one recognized vascular TRPV1. TRPV1 was expressed in blood vessels in the skeletal muscle, mesenteric and skin tissues, as well as in the aorta and carotid arteries. TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV1 in the same tissue sections. Capsaicin (a TRPV1 agonist) evoked constrictions in skeletal muscle arteries and in the carotid artery, but had no effect on the femoral and mesenteric arteries or the aorta. In blood vessels, TRPV1 expression was detected in most of the large arteries, but there were striking differences at level of the small arteries. TRPV1 activity was suppressed in some isolated arteries. This tightly regulated expression and function suggests a physiological role for vascular TRPV1.     

Distinct binding properties distinguish LQ-type calmodulin-binding domains in cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels operate as transduction channels in photoreceptors and olfactory receptor neurons. Direct binding of cGMP or cAMP opens these channels which conduct a mixture of monovalent cations and Ca(2+). Upon activation, CNG channels generate intracellular Ca(2+) signals that play pivotal roles in the transduction cascades of the visual and olfactory systems. Channel activity is controlled by negative feedback mechanisms that involve Ca(2+)-calmodulin, for which all CNG channels possess binding sites. Here we compare the binding properties of the two LQ-type calmodulin binding sites, both of which are thought to be involved in channel regulation. They reside on the isoforms CNGB1 and CNGA4. The CNGB1 subunit is present in rod photoreceptors and olfactory receptor neurons. The CNGA4 subunit is only expressed in olfactory receptor neurons, and there are conflicting results as to its role in calmodulin-mediated feedback inhibition. We examined the interaction of Ca(2+)-calmodulin with two recombinant proteins that encompass either of the two LQ sites. Comparing binding properties, we found that the LQ site of CNGB1 binds Ca(2+)-calmodulin at 10-fold lower Ca(2+) levels than the LQ site of CNGA4. Our data provide biochemical evidence against a contribution of CNGA4 to feedback inhibition. In accordance with previous work on photoreceptor CNG channels, our results indicate that feedback control is the exclusive role of the B-subunits in photoreceptors and olfactory receptor neurons.     

SERCA pump isoform expression in endothelium of veins and arteries: Every endothelium is not the same

Endothelium from rat aorta expresses sarco/endoplasmic reticulum Ca₂₊(SERCA) pump gene SERCA3 where as the smooth muscle expresses SERCA2. This has led to the postulate that vascular endothelium expresses SERCA3. To test this postulate, we examined the SERCA2 and SERCA3 mRNA expression in endothelium and smooth muscle dissected from coronary artery, coronary vein, aorta and vena cava of pig. Smooth muscle from all arteries and veins expressed only the SERCA2 mRNA. Endothelium from coronary artery, coronary vein and aorta expressed both SERCA2 and SERCA3 mRNA but the endothelium from vena cava did not express SERCA3 mRNA although it expressed SERCA2. These observations support the postulate that vascular endothelium expresses SERCA3 but the affirmation is equivocal because vena cava endothelium does not express SERCA3. (Mol Cell Biochem 000: 000-000, 1999)     

(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles

We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle.     

Functional activity of photoreceptor cyclic nucleotide-gated channels is dependent on the integrity of cholesterol- and sphingolipid-enriched membrane domains

Rod and cone photoreceptor cyclic nucleotide-gated (CNG) channels play pivotal roles in phototransduction. This work investigates the functional significance of photoreceptor CNG channel association with membrane microdomains enriched in raft lipids, cholesterol and sphingolipids. The primary subunits of cone and rod CNG channels, CNGA3 and CNGA1, respectively, were heterologously expressed in HEK 293 cells, and channel activity was determined by ratiometric measurement of [Ca (2+)] i in response to cyclic guanosine monophosphate (cGMP) stimulation. CNGA3 was found to be largely insoluble following Triton X-100 extraction and cofractionationed with biochemically isolated membrane domains enriched in caveolin-1. Cofractionation of both natively expressed CNGA3 and CNGB1 (the modulatory subunit of the rod CNG channel) with the low buoyant density, caveolin-1-enriched membranes was also confirmed in mouse retinas. The functional significance of this association was established by the observed negative effects of depletion of raft lipids on the channel activity. Treatment with the cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), significantly inhibited CNGA3 and CNGA1 activation in response to cGMP stimulation. MCD treatment lowered cellular cholesterol levels by approximately 45% without altering fatty acid composition, suggesting that the inhibition of channel activity by MCD treatment is not due to perturbation of other membrane lipids. Treatment with the sphingolipid biosynthesis inhibitor myriocin resulted in impaired activation and cytosolic redistribution of CNGA3, suggesting that the integrity of the membrane domains is critical for the channel cellular processing and plasma membrane localization. This study demonstrates the association of photoreceptor CNG channels with membrane domains enriched in raft lipids and indicates, for the first time, that raft lipids modulate the plasma membrane localization and functional activity of photoreceptor CNG channels.     

Endothelial K(+) channel lacks the Ca(2+) sensitivity-regulating beta subunit.

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.     

Inositol 1,4,5-trisphosphate receptor subtypes are differentially distributed between smooth muscle and endothelial layers of rat arteries

In blood vessels, the ability to control vascular tone depends on extracellular calcium entry and the release of calcium from inositol 1,4,5-trisphosphate receptor (IP3R)-gated stores located in both the endothelial and smooth muscle cells of the vascular wall. Therefore, we examined mRNA expression and protein distribution of IP3R subtypes in intact aorta, basilar and mesenteric arteries of the rat. IP3R1 mRNA was predominantly expressed in all three arteries. Immunohistochemistry showed that IP3R1 was present in both the muscle and endothelial cell layers, while IP3R2 and IP3R3 were largely restricted to the endothelium. Weak expression of IP3R2 was observed in the smooth muscle of the basilar artery. Co-localisation studies of IP3R subtypes with known cellular elements showed no association of any of the three subtypes with the endothelial cell plasma membrane, but a close association between the subtypes and actin filaments was observed in all cell layers. IP3R2 was found to be present near the endothelial cell nucleus. We are the first to demonstrate differential IP3R subtype distribution between the cell layers of the intact vascular wall and hypothesise that this may underlie the diversity of IP3R-dependent responses, such as vasoconstriction, vasodilation and vasomotion, displayed by arteries.     

Pulmonary expression of voltage-gated calcium channels: special reference to sensory airway receptors

Studying depolarisation induced calcium entry in our recently developed in situ lung slice model for molecular live cell imaging of selectively visualised pulmonary neuroepithelial bodies (NEBs), exemplified the need for information on the localisation of voltage-gated calcium channels (Ca(v)) in lungs in general, and related to sensory airway receptors more specifically. The present study therefore aimed at identifying the expression pattern of all major classes and subtypes of Ca(v) channels, using multiple immunostaining of rat lung cryosections. Ca(v) channel antibodies were combined with antibodies that selectively label NEBs, nerve fibre populations, smooth muscle, endothelium and Clara cells. Ca(v)2.1 (P/Q-type) was the only Ca(v) channel expressed in NEB cell membranes, and appeared to be restricted to the apical membrane of the slender NEB cell processes that reach the airway lumen. Subpopulations of the vagal but not the spinal sensory nerve fibres that contact NEBs showed immunoreactivity (IR) for Ca(v)1.2 (L-type) and Ca(v)2.1. Ca(v)2.3 (R-type) was selectively expressed by the so-called Clara-like cells that cover NEBs only, and appears to be a unique marker to discriminate this epithelial cell type from the much more extensive group of Clara cells in rat airways. The laminar nerve endings of smooth muscle-associated airway receptors (SMARs) revealed IR for both Ca(v)2.1 and Ca(v)2.2 (N-type). More generally, Ca(v)1.2 was seen to be expressed in vascular smooth muscle, Ca(v)2.3 and Ca(v)3.1 (T-type) in bronchial smooth muscle, Ca(v)3.1 and Ca(v)3.2 (T-type) in endothelial cells, and Ca(v)1.3 (L-type) in a limited number of epithelial cells. In conclusion, the present immunocytochemical study has demonstrated that the various subtypes of Ca(v) channels have distinct expression patterns in rat lungs. Special focus on morphologically/neurochemically characterised sensory airway receptors learned us that both NEBs and SMARs present Ca(v) channels. Knowledge of the identification and localisation of Ca(v) channels in airway receptors and surrounding tissues provides a solid basis for interpretation of the calcium mediated activation studied in our ex vivo lung slice model.     

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.     

NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.     

Dequalinium: a novel,high-affinity blocker of CNGA1 channels

Cyclic nucleotide-gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca(2+)-activated K(+) channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K(1/2) approximately 190 nM and CNGA1+CNGB1 channels with a K(1/2) approximately 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zdelta approximately 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.     

The expression, activity and localisation of the secretory pathway Ca2+ -ATPase (SPCA1) in different mammalian tissues

The distribution of the secretory pathway Ca2+ -ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney. SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca2+ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca2+ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.     

Subcellular origin of the oxalate- or inorganic phosphate-stimulated Ca2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin

Saponin, a cell-skinning reagent which perforates the cell membrane via its specific interaction with plasmalemmal cholesterol, was used to identify the subcellular origin of ATP-dependent Ca2+ accumulation in the presence and absence of inorganic phosphate and oxalate by microsomal fractions isolated from rat vas deferens and dog aorta. The purified plasma membranes from rat gastric fundus muscle, which elicit the stimulation of ATP-dependent Ca2+ accumulation by inorganic phosphate but not by oxalate, were used as a control reference. Saponin at concentrations effective for skinning smooth muscle fibres (10-50 micrograms/ml) inhibited Ca2+ binding in the absence of ATP to a similar extent in all fractions, but the inhibition of ATP-dependent Ca2+ accumulation was more pronounced in dog aorta microsomes and rat gastric fundus muscle plasma membranes than in rat vas deferens microsomes. The resistance of phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation to inhibition by saponin was much greater in rat vas deferens than in dog aorta microsomes. Our results suggest that phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation also occurs in plasma membrane vesicles isolated from smooth muscle and is by no means an unique property of endoplasmic reticulum.     

Molecular basis of ATP-sensitive K+ channels in rat vascular smooth muscles

ATP-sensitive K+ (K(ATP)) channels couple metabolic changes to membrane excitability in vascular smooth muscle cells (SMCs). While the electrophysiological properties of K(ATP) channels have been examined, little is known about the molecular basis of K(ATP) complex in vascular SMCs. We identified and cloned four K(ATP) subunit genes from rat mesenteric artery, namely rvKir6.1, rvKir6.2, rvKirSUR1, and rvSUR2B. These clones showed over 99.6% amino acid sequence identity with other previously reported isoforms. The mRNA expression patterns of the K(ATP) subunits varied among rat aorta, mesenteric artery, pulmonary artery, tail artery, hepatic artery, and portal vein. Heterologous co-expression of rvKir6.1 and rvSUR2B yielded functional K(ATP) channels that were inhibited by glibenclamide, and opened by pinacidil. Our results for the first time reported the expression of four K(ATP) subunits in same vascular tissues, unmasking the diversity of native K(ATP) channels in vascular SMCs.