Cholesterol enrichment increases basal and agonist-stimulated calcium influx in rat vascular smooth muscle cells.

The effect of cholesterol enrichment on vascular smooth muscle cell (VSMC) calcium homeostasis was studied by evaluating calcium uptake, efflux, and intracellular content in cultured VSMC derived from the rat pulmonary artery. Incubation of VSMC with liposomes consisting of free cholesterol (FC) and phospholipid (2:1 molar ratio, 1 mg FC/ml medium) for 24 h resulted in a 69 +/- 19% increase (P less than 0.01; n = 10) in FC which was associated with a 73 +/- 11% increase (P less than 0.005; n = 10) in intracellular calcium content as assessed by isotopic equilibrium with 45Ca2+ and a 65 +/- 11% increase (P less than 0.024; n = 3) as assessed by atomic absorption spectroscopy. Cholesterol enrichment caused a marked increase in the unidirectional calcium uptake rate from 0.026 +/- 0.03 to 0.158 +/- 0.022 nmol calcium/s per mg protein (P less than 0.01; n = 3), but had no effect on calcium efflux. Nifedipine (1 microM) reduced (P less than 0.05; n = 6) the effect of cholesterol enrichment on unidirectional calcium uptake by 78 +/- 16%; and verapamil (10 microM), diltiazem (1 microM), and nifedipine (1 microM) each significantly inhibited the effect of cholesterol enrichment on intracellular calcium accumulation. Exposure of cholesterol-enriched VSMC to cholesterol-poor liposomes for 24 h returned both FC and calcium contents to control levels. Serum- and serotonin-stimulated calcium uptakes were potentiated 3.7- and 1.7-fold, respectively, in cholesterol-enriched VSMC, whereas endothelin, vasopressin, and thrombin-stimulated calcium uptakes were not affected. We conclude that VSMC FC content plays a role in regulating cellular calcium homeostasis, both under basal conditions and in response to selected agonists.

IBC's conference on pulmonary hypertension: new understanding for therapeutic development.

The International Business Communications conference on 'Pulmonary Hypertension: New Understanding for Therapeutic Development' was held in San Diego, CA, USA. Approximately 100 basic scientists and clinicians from academia and industry participated in this highly interactive two-day symposium on pulmonary hypertension (PHT). Topics discussed included current clinical therapies and novel therapeutic strategies, e.g., nitric oxide, phosphodiesterase (PDE) inhibition, prostacyclin synthase expression, endothelin receptor antagonism and elastase inhibition.

KCa channel antagonists reduce NO donor-mediated relaxation of vascular and tracheal smooth muscle.

Electrophysiological studies suggest that activation of large-conductance Ca-activated K channels (KCa) with nitric oxide (NO) causes hyperpolarization and relaxation of smooth muscle. We determined whether KCa blockers decreased relaxation to the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydonimine-hydrochloride (SIN-1) in isolated segments from main pulmonary artery (MPA), its left branch (LPA), aorta (Ao), carotid artery (CA), and trachea (Tr). NO donors caused concentration-dependent relaxation of tissues precontracted with histamine whereas the inactive carrier molecule C88-3934 was without effect. The rank order profiles of SNAP and SIN-1 sensitivity were CA = Ao = MPA > LPA = Tr. Compared with histamine, 80 mM KCl precontraction caused variable reductions in tissue sensitivity and maximum relaxation to SNAP. The KCa antagonists charybdotoxin, iberiotoxin, and tetraethylammonium decreased sensitivity to SNAP and SIN-1 2- to 11-fold in MPA, LPA, and Tr, with variable shifts in Ao and CA. The effect of iberiotoxin was not altered by removing the endothelium or epithelium. Furthermore, charybdotoxin or iberiotoxin did not alter basal or SNAP-stimulated guanosine 3',5'-cyclic monophosphate content. Glibenclamide, noxiustoxin, and leiurotoxin I, antagonists of ATP-dependent, delayed rectifier, and small-conductance KCa channels, respectively, had no effect. In conclusion, antagonists of KCa decrease NO donor-mediated relaxation of pulmonary arterial and tracheal smooth muscle.

Acute exposure to cholesterol increases arterial nitroprusside- and endothelium-mediated relaxation.

The effect of cholesterol enrichment on arterial relaxation was studied by evaluating sodium nitroprusside (SNP)- and endothelium-mediated relaxation of isolated rabbit carotid artery. Arterial segments were perfused in vitro (4 h) with cholesterol-rich liposomes consisting of free cholesterol (FC) and phospholipid (PL) in a 2:1 molar ratio. Ring segments from arteries exposed to cholesterol-rich liposomes exhibited a 60% increase (P < 0.01) in FC content without affecting PL content. Cholesterol-enrichment was associated with a twofold increase (r = 0.92, P < 0.05) in acetylcholine- and A23187-induced endothelium-mediated relaxation. Bioassay of endothelium-derived relaxing factor(s) (EDRF) after cholesterol exposure indicated that EDRF half-life and/or release increased (P < 0.05) threefold. A trend (P = 0.07) toward increased smooth muscle cell sensitivity to EDRF after cholesterol enrichment was also observed. Cholesterol enrichment increased (P < 0.05) sensitivity to SNP 12-fold, and this difference was further augmented (P < 0.01) twofold with endothelium removal. Cholesterol enrichment had no effect on relaxation to N2,2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate. These data indicate that acute cholesterol enrichment increases EDRF activity from arterial endothelium and increases smooth muscle responses to both EDRF and SNP.

Excess membrane cholesterol alters calcium channels in arterial smooth muscle.

We studied the effects of cholesterol enrichment on arterial function by evaluating its effects on 45Ca2+ uptake and tension development in the carotid artery of the rabbit. Arterial segments were enriched with cholesterol in vitro, using media containing liposomes composed of free (unesterified) cholesterol (FC) and phospholipid (PL) in a 2:1 molar ratio. Control segments were simultaneously perfused with 0.5:1 liposomal medium to compare the possible effects of PL. Rings from these arteries were then tested for basal and activated Ca2+ uptake and for contractile responses to norepinephrine (NE) and KCl. We found elevated 45Ca2+ uptake under basal and NE-activated conditions along with an increased contractile sensitivity (4-fold) to NE. These alterations correlated with a 78% increase in the FC/PL ratio reflecting cholesterol enrichment of cellular membranes. Cholesterol enrichment did not alter resting or maximal tensions, K+-activated Ca2+ uptake, or contractile sensitivity to K+. Pretreatment with 1 microM diltiazem abolished the cholesterol-induced increase in basal as well as NE-activated 45Ca2+ uptake but had no effect on either uptake in control vessels. These studies suggest that excess membrane cholesterol selectively increases NE contractile sensitivity by increasing basal or NE-activated Ca2+ influx (or both) as a result of fundamental alteration in the calcium channels in arterial smooth muscle cell membrane.

Stretching increases calcium influx and efflux in cultured pulmonary arterial smooth muscle cells.

To determine the effect of a single static stretch on calcium fluxes in cultured pulmonary arterial smooth muscle cells (PASMC), calcium influx and efflux were evaluated in PASMC on a collagen-coated silicone membrane using 45Ca2+ as a tracer. A single 20% linear stretch of the silicone membrane of 1 min in duration increased calcium uptake by 71%. This effect was partially inhibited by verapamil or gadolinium, but was not altered by staurosporine, pertussis toxin, or removal of extracellular sodium. Stretch-stimulated calcium uptake attenuated over time, such that uptake during the last minute of a 5-min sustained stretch was 46% of that during the first minute of stretch. A single 20% stretch sustained for 6 min caused a 47% increase in calcium efflux, the magnitude of which was linearly related to the degree of cell stretch. Gadolinium and removal of extracellular calcium each partially inhibited stretch-induced calcium efflux. We conclude that a single static stretch of PASMC causes increases in both calcium influx and efflux. Stretch-stimulated calcium influx does not require sodium influx and is mediated in part by a pathway sensitive to both gadolinium and verapamil. Stretch-stimulated calcium efflux is due to both calcium influx via a gadolinium-sensitive pathway and mobilization of intracellular stores. Because calcium is a key cellular second messenger, these effects of stretch on cellular calcium handling may play a role in the regulation of vascular smooth muscle cell phenotype and function.

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells.

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.

Cholesterol increases the L-type voltage-sensitive calcium channel current in arterial smooth muscle cells.

To determine whether membrane free cholesterol affects calcium currents in vascular smooth muscle cells, whole-cell patch clamp recordings were made before and after cholesterol enrichment of cells by exposure to cholesterol-rich liposomes. Exposure to cholesterol-rich liposomes resulted in a gradual increase in the L-type current over 20 hours and a plateau (73 +/- 7% increase over basal) between 20 and 32 hours. This effect was associated with a rightward shift in the inactivation potential and a decrease in the sensitivity to (-)-PN-202-791, a dihydropyridine antagonist. There was no change in the maximum L-type current stimulated by (+)-PN-202-791, a dihydropyridine agonist. Liposome exposure caused a small, transient increase in the T-type current (peak effect, 20 minutes). We conclude that membrane cholesterol has important effects on the L-type calcium current in vascular smooth muscle cells, which is most likely due to an alteration in channel functional state rather than an increase in channel expression.

Chronic hypoxia increases staurosporine sensitivity of pulmonary artery smooth muscle to endothelin-1.

Endothelins (ET) have been implicated in the pathogenesis of hypoxia-induced pulmonary hypertension. We evaluated the contribution of protein kinase C (PKC) to the ET-1 response of isolated endothelium-denuded extralobar pulmonary artery (PA) from rats exposed to chronic hypoxia (10% O2-90% N2, 1 ATM, 14 days or 28 days) or air. Hypoxia increased hematocrit (Hct [% above air control]: at 14 days, by 28+/-2%; after 28 days to 33+/-2%) and the mass ratio of right ventricle over left ventricle plus septum (RV/LV+S [% above air control]: at 14 days, by 54+/-1%; after 28 days to 114+/-13%), an index of right ventricle hypertrophy. Hypoxic exposure for 14 days and 28 days decreased PA sensitivity to ET-1 (change in EC50: 14 days, four-fold; 28 days, two-fold vs. air controls) and transiently decreased the magnitude of maximum ET-1-induced contraction (Emax [% decrease from control]: 14 days, 53+/-6%; 28 days, 23+/-6%). Staurosporine, a PKC inhibitor, decreased ET-1 sensitivity of PA from 0, 14, and 28 days air-exposed rats by four- to nine-fold without affecting Emax. However, staurosporine markedly decreased hypoxic PA sensitivity to ET-1 (change in EC50: 14 days, 1700-fold; 28 days, 55-fold vs. hypoxic controls) and decreased Emax (% decrease from hypoxic control: 14 days, 38+/-6%; 28 days, 59+/-7%). In conclusion, hypoxic exposure time-dependently varies the responsiveness of PA smooth muscle to ET-1 and may modulate the contribution of PKC activation.

Stretch increases inositol trisphosphate and inositol tetrakisphosphate in cultured pulmonary vascular smooth muscle cells.

There are no reports of the effect of stretch on inositol phosphates in smooth muscle. Phosphoinositide and inositol phosphate metabolism was studied in cultured rat vascular smooth muscle cells subjected to stretching. The masses of inositol trisphosphate and tetrakisphosphate increased (+34 +/- 7% and +58 +/- 12%, respectively; p less than 0.001) after 25 s of a single 20% stretch and had returned to control levels by 45 s; phosphatidylinositol, phosphatidylinositol phosphate and bisphosphate did not change. Repetitive stretch did not alter the masses of any of the compounds. A single stretch also increased 45Ca2+ efflux (+52 +/- 5%, p less than 0.01). These data suggest that stretch of cultured vascular smooth muscle can elicit a rapid, short-lived increase in inositol phosphates, which may subsequently affect Ca2+.

Functional comparison of endothelin receptors in human and rat pulmonary artery smooth muscle.

The receptors mediating arterial smooth muscle contraction to endothelins (ET) differ among species and origin of vascular bed. We characterized ET receptors mediating contraction of endothelium-denuded human intralobar pulmonary artery (hIPA) and rat intralobar (rIPA) and extralobar left branch (rLPA) pulmonary artery with ET-1, ET-2, ET-3, sarafotoxin S6c, sarafotoxin S6b, and ET receptor antagonists in vitro. Rat aorta was studied for comparison. Each vascular segment showed concentration-dependent contraction with a rank order sensitivity (pD2) profile of ET-1 > or = ET-2 = sarafotoxin S6b > ET-3. Maximum contraction to ET-1 was greater than to sarafotoxin S6c in all preparations. Responses of rIPA and rLPA to sarafotoxin S6c were conspicuous when compared with hIPA or aorta. The ET(A) receptor blockers BQ-123 and BMS-182874 competitively antagonized ET-1 responses of hIPA and aorta, but not rLPA. The ET(B) receptor antagonist BQ-788 attenuated contractions of rIPA and rLPA to ET-3 and sarafotoxin S6c, respectively. In conclusion, ET(B)-mediated contraction of endothelium-denuded conduit pulmonary arteries varies among species and may contribute more to contraction of rIPA and rLPA than of hIPA and aorta, although maximum ET(B)-mediated contraction is smaller than that mediated by the ET(A) receptor.

Hypoxic exposure time dependently modulates endothelin-induced contraction of pulmonary artery smooth muscle.

Endothelins (ETs) have been implicated in the pathogenesis of hypoxia-induced pulmonary hypertension. We determined whether hypoxic exposure of rats (10% O2-90% N2, 1 atm, 1-48 days) altered contraction to ET in isolated segments of endothelium-denuded extralobar branch pulmonary artery (PA) and aorta. Hypoxic exposure increased hematocrit, right ventricular hypertrophy, and ET-1 plasma concentration. Hypoxia also caused a sustained decrease in PA but not in aorta sensitivity to ET-1. In comparison, hypoxic exposure throughout 12 days decreased time dependently the maximum contraction of PA to ET-1, BaCl2, and KCl. The hypoxia-induced decrease in maximum contraction of PA to ET-1 returned toward normal levels by 21 days and approximated control levels by 48 days. After 14 days of hypoxia, right ventricular hypertrophy correlated with decreased sensitivity of PA to ET-1. After 21 days of hypoxia, PA sensitivity to ET-2 and ET-3 was decreased, and sarafotoxin S6c-induced contraction was abolished. In conclusion, hypoxic exposure time dependently modulates the responsiveness of PA smooth muscle to ETs, BaCl2, and KCl. The hypoxia-induced changes in tissue responsiveness to ET-1 may be associated with increased plasma concentrations of this peptide.

ZD1611, an orally active endothelin-A receptor antagonist, prevents chronic hypoxia-induced pulmonary hypertension in the rat.

Endothelin-1 (ET-1) is a potent vasoconstrictor and comitogen implicated in the pathogenesis of pulmonary hypertension (PH). We evaluated the effects of an ET(A)receptor-selective antagonist, ZD1611, on hypoxia-induced PH in rats. <> and <> paradigms were established in which rats were administered placebo or ZD1611 (1-3 mg/kg, q.i.dpo) concomitant with hypoxic exposure (10% O(2)1 ATM) for 14 days or beginning after 7-day hypoxic exposure for 21 days. Compared with normoxic controls, hypoxic exposure plus placebo increased (P<0.05) hematocrit, mass ratio of right ventricle over left ventricle plus septum (RV/LV+S), and right intraventricular peak systolic pressure (RVSP). These latter two effects were decreased (P<0.05) by ZD1611 in both experimental paradigms [RV/LV+S(%)::RVSP(%); prophylactic, 14::32; therapeutic, 28::37]. Hypoxic exposure did not change mean systemic arterial pressure (MSAP). ZD1611 did not affect MSAP, plasma ET-1 concentrations, or blood gases measured when rats respired room air. In mechanistic studies, ZD1611 decreased (P<0.01) smooth muscle hypertrophy of small pulmonary arteries and abolished hypoxia-induced decreases in sensitivity and maximum contraction to ET-1 in isolated extralobar branch pulmonary artery. In conclusion, the ET(A)receptor-selective antagonist, ZD1611, attenuates hypoxia-induced PH in the rat.

Pharmacological characterization of ZD6021: a novel, orally active antagonist of the tachykinin receptors.

The tachykinins, substance P, neurokinin A, and neurokinin B, have been implicated in many diseases. The present study evaluated the pharmacological properties of a novel tachykinin antagonist ZD6021 [3-cyano-N-((2S)-2-(3,4-dichlorophenyl)-4-[4-[2-(methyl-(S)-sulfinyl)-phenyl]piperidino]butyl)-N-methyl-]-napthamide]. The affinity (K(i)) of ZD6021 for the cloned human neurokinin (NK)1, NK2, and NK3 receptors was 0.12 +/- 0.01, 0.64 +/- 0.08, and 74 +/- 13 nM, respectively. Mucin secretion by Chinese hamster ovary cells transfected with the human NK1 receptor was dose dependently inhibited by ZD6021: pIC(50) = 7.6 +/- 0.1. For NK1 and NK2 receptors, the agonist concentration-response curves using isolated tissues were displaced rightward in the presence of ZD6021: rabbit pulmonary artery, pA2 = 8.7 and 8.5; human pulmonary artery and bronchus, pKB = 8.9 +/- 0.4 and 7.5 +/- 0.2, at 10(-7) M, respectively. Senktide-induced contractions of isolated guinea pig ileum were also blocked by low concentrations of ZD6021. Oral administration of ZD6021 to guinea pigs dose dependently attenuated tracheal extravasation of plasma proteins induced by the NK1 receptor agonist Ac-[Arg6,Sar9,Met(O2)11]-SP(6-11), ED50 = 0.8 micromol/kg, and bronchoconstriction, elicited by the NK2 receptor agonist [beta-Ala8]-NKA(4-10), ED50 = 20 micromol/kg. Potency was unaffected by feeding. After oral administration of ZD6021, the time to peak activity was 150 min for the NK1 receptor and 60 min for the NK2 receptor with pharmacodynamic half-lives of 280 and 458 min, respectively. These data indicate that ZD6021 is a potent, orally active antagonist of all three tachykinin receptors. This compound may be useful for future studies of tachykinin-related pathology such as asthma.

The discovery of non-basic atrial natriuretic peptide clearance receptor antagonists. Part 1.

The cyclic peptide ANP 4-23 and the linear peptide analogue AP-811 have been shown to be selective ANP-CR antagonists. Via alanine scanning and truncation studies we sought to determine which residues in these molecules were important in their binding to the clearance receptor and the relationship between these two molecules. These studies show that several modifications to these compounds are possible which improve physical properties of these molecules while retaining high affinity for the ANP-CR.