Direct regulation of smooth muscle contractile elements by second messengers

The effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and phorbol 12,13 dibutyrate (PDBu) on the Ca2+ sensitivity of the contractile elements in the rat mesenteric artery were investigated, using a method of permeabilizing smooth muscle with Staphylococcal alpha-toxin. Both cAMP and cGMP relaxed the permeabilized rat mesenteric artery at the intracellular Ca2+ concentrations [( Ca2+]i) held constant with Ca2+ EGTA buffer and Ca2+ ionophore, ionomycin. In addition, forskolin and sodium nitroprusside which activate adenylate and guanylate cyclases, respectively, also induced relaxation at a fixed [Ca2+]i. In contrast PDBu which stimulates protein kinase C caused an increase in force at a constant [Ca2+]i which could be partially reversed by cAMP or cGMP. These results indicate that second messengers exert direct control over smooth muscle Ca2+ sensitivity of the contractile elements, which is of physiologic and pharmacologic importance.     

Diversity of K+ channels in circular smooth muscle of opossum lower esophageal sphincter

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.     

Nicorandil reduces the basal level of cytosolic free calcium in single guinea pig ventricular myocytes.

Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM approximately 1 mM) reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membrane permeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM) nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 microM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 +/- 80 ms to 153 +/- 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (approximately 2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.     

K+-induced alterations in airway muscle responsiveness to electrical field stimulation

We investigated possible pre- and postsynaptic effects of K+-induced depolarization on ferret tracheal smooth muscle (TSM) responsiveness to cholinergic stimulation. To assess electromechanical activity, cell membrane potential (Em) and tension (Tm) were simultaneously recorded in buffer containing 6, 12, 18, or 24 mM K+ before and after electrical field stimulation (EFS) or exogenous acetylcholine (ACh). In 6 mM K+, Em was -58.1 +/- 1.0 mV (mean +/- SE). In 12 mM K+, Em was depolarized to -52.3 +/- 0.9 mV, basal Tm did not change, and both excitatory junctional potentials and contractile responses to EFS at short stimulus duration were larger than in 6 mM K+. No such potentiation occurred at a higher K+, although resting Em and Tm increased progressively above 12 mM K+. The sensitivity of ferret TSM to exogenous ACh appeared unaffected by K+. To determine whether the hyperresponsiveness in 12 mM K+ was due, in part, to augmented ACh release from intramural airway nerves, experiments were done using TSM preparations incubated with [3H]choline to measure [3H]ACh release at rest and during EFS. Although resting [3H]ACh release increased progressively in higher K+, release evoked by EFS was maximal in 12 mM K+ and declined in higher concentrations. We conclude that small elevations in the extracellular K+ concentration augment responsiveness of the airways, by increasing the release of ACh both at rest and during EFS from intramural cholinergic nerve terminals. Larger increases in K+ appear to be inhibitory, possibly due to voltage-dependent effects that occur both pre- and postsynaptically.     

Serotonin depolarizes the membrane potential in rat mesenteric artery myocytes by decreasing voltage-gated K+ currents

We hypothesized that voltage-gated K+ (Kv) currents regulate the resting membrane potential (Em), and that serotonin (5-HT) causes Em depolarization by reducing Kv currents in rat mesenteric artery smooth muscle cells (MASMCs). The resting Em was about -40 mV in the nystatin-perforated patch configuration, and the inhibition of Kv currents by 4-aminopyridine caused marked Em depolarization. The inhibition of Ca2+-activated K+ (KCa) currents had no effect on Em. 5-HT (1 microM) depolarized Em by approximately 11 mV and reduced the Kv currents to approximately 63% of the control at -20 mV. Similar 5-HT effects were observed with the conventional whole-cell configuration with a weak Ca2+ buffer in the pipette solution, but not with a strong Ca2+ buffer. In the presence of tetraethylammonium (1mM), 5-HT caused Em depolarization similar to the control condition. These results indicate that the resting Em is largely under the regulation of Kv currents in rat MASMCs, and that 5-HT depolarizes Em by reducing Kv currents in a [Ca2+]i-dependent manner.     

Cyclic nucleotide function in trachealis muscle of dogs with and without airway hyperresponsiveness

We examined basal adenosine 3',5'-cyclic monophosphate (cAMP) levels, isoproterenol (ISO)-stimulated cAMP responses, basal cAMP, and guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (PDE) activities and protein-kinase (PK) activities in trachealis muscle from five Basenji-greyhound (BG) and four greyhound dogs to determine whether the inverse relationship between in vivo and in vitro airway responsiveness could be due to altered cyclic nucleotide metabolism. Basal cAMP levels were not significantly different (PNS) in muscle from BG (11.6 +/- 0.53 pmol/mg protein) and greyhound dogs (10.30 +/- 1.60 pmol/mg protein). The cAMP responses to stimulation with ISO were enhanced in BG compared with greyhound dogs. The low Michaelis constant (1) for Km-cAMP PDE activity (Km = 0.63 microM) was significantly less (P less than 0.005) in BG dogs (1.54 +/- 0.28 pmol.min-1.mg protein-1) than greyhounds (11.76 +/- 2.48). Endogenously active PK activity was significantly greater (P less than 0.005) in BG (54.74 +/- 5.39 pmol.min-1.mg protein-1) than in greyhound dogs (15.50 +/0 2.20). Increases in PK activity with 5 microM cAMP added were not significantly different between BG (14.79 +/- 6.00) and greyhound dogs (7.04 +/- 2.14). Approximately 90% of both endogenous PK activity and cAMP-activated PK activity in BG and greyhound dogs was inhibited by a cAMP-dependent PK inhibitor (PKI'). These data suggest that decreased cyclic nucleotide degradation due to decreased cyclic nucleotide PDE activity with increased PK could account for the in vitro hyporesponsiveness of airway smooth muscle in BG dogs as a protective adaptive mechanism.     

Regulation of Ca2+ handling by phosphorylation status in mouse fast- and slow-twitch skeletal muscle fibers

The effects ofphosphorylation status on Ca²⁺release and Ca²⁺ removal werestudied in fast-twitch flexor digitorum brevis and slow-twitch soleusskeletal muscle fibers enzymatically isolated from wild-type andphospholamban knockout (PLBko) mice. In all fibers the adenosine3',5'-cyclic monophosphate-dependent protein kinase (PKA)inhibitor H-89 decreased the peak amplitude of the intracellularCa²⁺ concentration([Ca²⁺]) transient fora single action potential, and the PKA activator dibutyryl adenosine3',5'-cyclic monophosphate (DBcAMP) reversed this effect,indicating modulation of Ca²⁺release by phosphorylation status in all fibers. H-89 decreased thedecay rate constant of the[Ca²⁺] transient andDBcAMP reversed this effect only in phospholamban-expressing fibers(wild-type soleus), indicating modulation ofCa²⁺ removal only in the presenceof phospholamban. A high basal level of PKA phosphorylation in soleusfibers maintained under our control conditions was indicated bythe lack of effect of direct application of DBcAMP onCa²⁺ release orCa²⁺ removal in wild-type or PLBkosoleus fibers and was confirmed by analysis of phospholamban fromwild-type soleus fibers.

     

Dual effects of lysophosphatidic acid on human airway smooth muscle cell proliferation and survival

Lysophosphatidic acid (LPA) is a phospholipid growth mediator found in serum at 2-20 microM. In many cell types, including human airway smooth muscle (HASM) cells, LPA-induced proliferation occurs at 10-100 microM LPA. At these concentrations LPA forms Ca2+ precipitates. The potential involvement of Ca2+ and Ca2+ LPA precipitates in LPA-induced HASM cell mitogenesis was investigated. In the absence of extracellular Ca2+, 10 and 30 microM LPA stimulated HASM cell mitogenesis. However, with 100 microM LPA in the absence of extracellular Ca2+, HASM cells exhibited a profound shape change and loss of viability, determined to be apoptosis by both DNA staining and assessment of cytosolic nucleosomal reactivity. A bioassay based on the adenosine 3':5'-cyclic monophosphate response of C62B rat glioma cells was used to measure the bioactivity of LPA solutions prepared in Ca2+ free and Ca2+ containing medium. After 24 h, a 100 microM LPA solution in Ca2+ free medium contained markedly greater bioactivity than a 100 microM LPA solution made in Ca2+ containing medium. In summary, formation of Ca2+ LPA precipitates decreases the amount of biologically active LPA in solution, and high concentrations of bioactive LPA achieved in Ca2+ free but not in Ca2+ containing medium induce apoptosis of HASM cells.     

8-Bromoguanosine 3':5'-cyclic monophosphate decreases intracellular free calcium concentrations in cultured vascular smooth muscle cells from rat aorta

The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br cGMP) on intracellular free calcium concentrations ([Ca2+]i) in cultured rat aortic vascular smooth muscle cells (VSMCs) loaded with fura-2 were recorded microfluorometrically. Irrespective of whether VSMCs were at rest (in 5 mM K+ PSS), under Ca2+ depletion (in Ca2+-free medium for 10 min) and K+ depolarization (in high K+ PSS), [Ca2+]i was actively reduced and reached a new and lower steady-state level with the application of 8-Br cGMP. This may be the first and direct evidence that cGMP, a putative mediator of various vasodilators, actively reduces [Ca2+]i in VSMCs.     

8-Bromo-cAMP decreases the Ca2+ sensitivity of airway smooth muscle contraction through a mechanism distinct from inhibition of Rho-kinase

To clarify whether cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) activation and Rho-kinase inhibition share a common mechanism to decrease the Ca2+ sensitivity of airway smooth muscle contraction, we examined the effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a stable cAMP analog, and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a Rho-kinase inhibitor, on carbachol (CCh)-, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-, 4beta-phorbol 12,13-dibutyrate (PDBu)-, and leukotriene D4 (LTD4)-induced Ca2+ sensitization in alpha-toxin-permeabilized rabbit tracheal and human bronchial smooth muscle. In rabbit trachea, CCh-induced smooth muscle contraction was inhibited by 8-BrcAMP and Y-27632 to a similar extent. However, GTPgammaS-induced smooth muscle contraction was resistant to 8-BrcAMP. In the presence of a saturating concentration of Y-27632, PDBu-induced smooth muscle contraction was completely reversed by 8-BrcAMP. Conversely, PDBu-induced smooth muscle contraction was resistant to Y-27632. In the presence of a saturating concentration of 8-BrcAMP, GTPgammaS-induced Ca2+ sensitization was also reversed by Y-27632. The 8-BrcAMP had no effect on the ATP-triggered contraction of tracheal smooth muscle that had been treated with calyculin A in rigor solutions. The 8-BrcAMP and Y-27632 additively accelerated the relaxation rate of PDBu- and GTPgammaS-treated smooth muscle under myosin light chain kinase-inhibited conditions. In human bronchus, LTD4-induced smooth muscle contraction was inhibited by both 8-BrcAMP and Y-27632. We conclude that cAMP/PKA-induced Ca2+ desensitization contains at least two mechanisms: 1) inhibition of the muscarinic receptor signaling upstream from Rho activation and 2) cAMP/PKA's preferential reversal of PKC-mediated Ca2+ sensitization in airway smooth muscle.     

Modulation of plasma membrane Ca2+ pump by membrane potential in cultured vascular smooth muscle cells

We examined the effect of membrane potential (Em) on the activity of the plasma membrane Ca2+ pump in cultured rat aortic smooth muscle cells (VSMCs). Inside-negative K+ diffusion potential higher or lower than the resting Em (-46 mV) was artificially imposed on VSMCs with various concentrations of extracellular K+ (K+o) and 1 microM valinomycin. We found that the recovery phase of the intracellular Ca2+ transient elicited with 1 microM ionomycin was accelerated by depolarizing Em, whereas it was retarded by hyperpolarizing Em. The rate of extracellular Na+ (Na+o)-independent 45Ca2+ efflux from VSMCs stimulated with 1 microM ionomycin increased almost linearly with a change in Em from -98 to -3 mV. This effect of Em was abolished by extracellularly added LaCl3 or a combination of high pH (pH 8.8) and high Mg2+ (20 mM), conditions that presumably inhibit the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., & Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Intracellular contents of Na+ and K+ and intracellular pH, on the other hand, were not influenced by the change in Em under the conditions used. These results indicate that alteration in Em can modulate the intracellular Ca2+ concentration in intact VSMCs by changing the rate of Ca2+ extrusion by the plasma membrane Ca2+ pump. The data strongly suggest that the plasma membrane Ca2+ pump in VSMCs is electrogenic.     

Long acting cAMP analogues enhance sulfate incorporation into matrix proteoglycans and suppress cell division of fetal rat chondrocytes in monolayer culture.

The relationship between replication and the synthesis of matrix sulfated proteoglycans was investigated with fetal rat chondrocytes grown in monolayer culture. The effect of N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP), adenosine 3', 5' cyclic monophosphate (cAMP), 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP), sodium butyrate and hydroxyurea was examined. Between 0.05 and 0.5 mM DBcAMP, a dose related inhibition of cell division and stimulation of [35SO=/4] incorporation into matrix proteoglycans was demonstrated. At the higher concentrations of DBcAMP, cell division was completely inhibited and the enhancement of [35SO=/4] incorporation into matrix proteoglycans ranged between 40 and 120% (P less than 0.01). Utilizing 14C-glucosamine and photometric determination of proteoglycans with Alcian Blue, it was demonstrated that the increase in sulfate incorporation reflected enhanced accumulation of extracellular matrix. The effects of DBcAMP were mimicked by 8 Br-cAMP, suggesting they were mediated by the adenylyl cyclase system. cAMP (0.05-0.5 mM), sodium butyrate (0.1-0.5 mM) and hydroxyurea (0.5-5 mM) partially or fully inhibited cell division, but either failed or only slightly enhanced sulfate incorporation. The enhanced sulfated proteoglycan deposition promoted by DBcAMP began 8 to 12 hours after serum stimulation, its onset occurred prior to thymidine incorporation and the effect persisted for 28 hours. Determination of cell volume demonstrated an increase in size of DBcAMP treated chondrocytes between 8 to 12 hours, coincident with the onset of increased sulfate incorporation. These results are consistent with a model where matrix sulfated proteoglycan deposition by chondrocytes is mediated by intracellular cAMP levels and occurs in the G1 phase of the cell cycle.     

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compounds that increase cAMP prevent ischemia-reperfusion pulmonary capillary injury.

This study evaluated the physiological effects of compounds that increase adenosine 3',5'-cyclic monophosphate (cAMP) on changes in pulmonary capillary permeability and vascular resistance induced by ischemia-reperfusion (I-R) in isolated blood-perfused rabbit lungs. cAMP was elevated by 1) beta-adrenergic stimulation with isoproterenol (ISO, 10(-5) M), 2) post-beta-receptor stimulation of adenylate cyclase with forskolin (FSK, 10(-5) M), 3) and dibutyryl cAMP (DBcAMP, 1 mM), a cAMP analogue. Vascular permeability was assessed by determining the capillary filtration coefficient (Kf,c), and capillary pressure was measured using the double occlusion technique. The total, arterial, and venous vascular resistances were calculated from measured pulmonary arterial, venous, and capillary pressures and blood flow. Reperfusion after 2 h of ischemia significantly (P less than 0.05) increased Kf,c (from 0.115 +/- 0.028 to 0.224 +/- 0.040 ml.min-1.cmH2O-1.100 g-1). These I-R-induced changes in capillary permeability were prevented when ISO, FSK, or DBcAMP was added to the perfusate at reperfusion (0.110 +/- 0.022 and 0.103 +/- 0.021, 0.123 +/- 0.029 and 0.164 +/- 0.024, and 0.153 +/- 0.030 and 0.170 +/- 0.027 ml.min-1.cmH2O-1.100 g-1, respectively). I-R significantly increased total, arterial, and venous vascular resistances. These increases in vascular resistance were also blocked by ISO, FSK, and DBcAMP. These data suggest that beta-adrenergic stimulation, post-beta-receptor activation of adenylate cyclase, and DBcAMP prevent the changes in pulmonary vascular permeability and vascular resistances caused by I-R in isolated rabbit lungs through a mechanism involving an increase in intracellular levels of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of renal gluconeogenesis by calcium ions, hormones and adenosine 3′:5′-cyclic monophosphate

1. The effect of Ca(2+), glucagon, adrenaline and adenosine 3':5'-cyclic monophosphate on gluconeogenesis by rat kidney-cortex slices was studied. 2. Glucose formation from a range of substrates, with the exception of glycerol, was increased by an increase in extracellular Ca(2+) concentration. 3. Hormones and adenosine 3':5'-cyclic monophosphate, at low Ca(2+) concentrations, stimulated glucose production from several substrates, but not from glycerol, fructose, malate or fumarate. 4. Hormonal stimulation was not detected in the absence of Ca(2+) or at 2.5mm-Ca(2+). 5. Ca(2+), hormones and adenosine 3':5'-cyclic monophosphate had no effect on phosphoenolpyruvate carboxylase activity. 6. It is proposed that Ca(2+) and adenosine 3':5'-cyclic monophosphate-mediated hormone action activate the same rate-limiting step in gluconeogenesis: this step is tentatively identified as the rate of transfer of substrates across the mitochondrial membrane.     

Effect of Bay K 8644 on tetraethylammonium-induced excitability of the rabbit pulmonary artery

The effect of Bay K 8644 on the electrical activity of the smooth muscle cells in the main pulmonary artery of the rabbit was examined. In normal physiological solution, the resting membrane potential was -56 +/- 0.6 mV, and the cells were electrically quiescent. Tetraethylammonium (5 mM) depolarized the membrane to about -45 mV, and electrical stimulation elicited action potentials. To suppress contractile responses and thereby facilitate sustained impalements, the muscle strips were bathed with a hypertonic solution containing sucrose. The mean amplitude of the tetraethylammonium-induced action potentials in the hypertonic solution was 35 +/- 0.9 mV. The action potentials were dependent upon the extracellular Ca2+ concentration and were abolished by diltiazem (10(-6) M). Spontaneous action potentials were occasionally generated in the presence of tetraethylammonium alone and could be induced by the further addition of Ba2+ (0.5 mM). The Ca2+ agonist Bay K 8644 (10(-8) to 10(-6) M) had no effect on the resting membrane potential or excitability in normal solution. However, in the hypertonic solution containing tetraethylammonium, Bay K 8644 caused a further depolarization and oscillatory potential changes, which were not prevented by tetrodotoxin. The oscillations were suppressed or abolished by diltiazem or nilvadipine. Thus, active responses can occur in the normally quiescent smooth muscle cells of the rabbit pulmonary artery when the outward K+ current(s) are suppressed.     

Hyperpolarizing effect of aminophylline, theophylline, and cAMP on rat diaphragm fibers

We studied the effect of aminophylline and theophylline (0.1-2 mM) on the resting membrane potential (Vm) of rat diaphragm fibers in vitro (25 degrees C). The main findings are the following. 1) Aminophylline and theophylline hyperpolarize the fibers in a dose-dependent manner. This effect is present with 0.1 and 0.25 mM of aminophylline and theophylline, respectively, and the maximum effect is reached with 1 mM of the drug (approximately 5-8 mV in comparison to the normal values). This effect is reversible by washing out the preparation with normal solution. 2) Dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP, 2 mM) produces a similar increment in the Vm. 3) The hyperpolarizing action observed in the presence of aminophylline, theophylline, and DBcAMP is suppressed by 5 X 10(-4) M ouabain or by lowering the bath temperature to 5 degrees C. These results suggest that the xanthines may directly or indirectly stimulate a Na-K pump. Two possibilities may be considered: 1) an electrogenic effect of the Na-K pump and 2) a reduction in the extracellular K+ concentration in the solution contacting the external side of the cell as a consequence of the activity of the Na-K pump. Alternative mechanisms such as a reduction in Na permeability or an increment in K permeability might collaborate in the hyperpolarizing effect of the drugs tested.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

Effect of exogenous cAMP and aminophylline on alveolar and lung liquid clearance in anesthetized sheep.

A substantial body of evidence indicates that active transport of ions is important in modulating the resolution process of pulmonary edema. The biochemical regulation of this ion transport mechanism is still under investigation. In this study we evaluated the effect of an adenosine 3',5'-cyclic monophosphate (cAMP) analogue [dibutyryl cAMP (DBcAMP)] and a phosphodiesterase inhibitor (aminophylline) given alone or together on lung liquid and protein clearance. To study lung liquid and protein clearance, we measured the removal of 100 ml of autologous serum from the air spaces of anesthetized and ventilated adult sheep. Either serum alone or serum mixed with 10(-3) M DBcAMP, 10(-3) M or 10(-5) M aminophylline, or 10(-3) M aminophylline plus 10(-3) M DBcAMP was instilled. After 4 h, the residual lung water was 73.5 +/- 8.7 ml when serum alone was instilled and 56.8 +/- 13.6 ml when aminophylline and DBcAMP were given together. Neither aminophylline nor DBcAMP alone increased lung liquid clearance. However, the increase in clearance cannot be explained by an increase in protein clearance or changes in the pulmonary hemodynamics. These data suggest that the cAMP second messenger system can stimulate lung liquid clearance in vivo.