Inhibition of phospholipase A2 attenuates functional hyperemia in the hamster cremaster muscle.

Arachidonic acid (AA) is the common precursor for several vasodilatory factors involved in the local control of blood flow. This study was designed to determine the role of phospholipase A2 (PLA2) and AA release in functional hyperemia in the hamster cremaster muscle. The muscle was prepared for in vivo microscopy and subjected to electrical field stimulation for 1 min. First- and second-order arterioles dilated in response from a mean diameter of 66 +/- 5 to 88 +/- 7 micrometer (n = 6). PLA2 was then inhibited with quinacrine (3 x 10(-6) M) for 60 min. PLA2 inhibition was verified by an attenuation of thrombin-induced vasodilation (2 U/ml). Quinacrine had no effect on resting arteriolar diameter but completely abolished functional hyperemia. Quinacrine also had no effect on dilation induced by superfusion of the preparation with 3 x 10(-6)-10(-5) M AA, 10(-6)-10(-4) M adenosine, or 10(-6)-10(-4) M sodium nitroprusside, ruling out nonspecific effects of quinacrine on smooth muscle contractility. These results indicate that functional hyperemia in the hamster cremaster muscle is dependent on PLA2 activation and the availability of AA.

Muscarinic receptors inhibit ATP-sensitive K+ channels in swine tracheal smooth muscle.

The patch-clamp technique was used to characterize a cromakalim-induced current and its regulation by muscarinic receptors in tracheal smooth muscle cells. Cromakalim (10 microM) activated a steady-state increase of 33-292 pA (91.2 +/- 8.8 pA, n = 43) in whole cell current, consistent with the activation of ATP-sensitive K+ (KATP) channels. Acetylcholine (10 nM-1 microM), added cumulatively, inhibited the cromakalim-induced current by 23.6 +/- 14.9 to 73.9 +/- 4.6%. This inhibition was blocked by pretreatment with atropine (1 microM). The cromakalim-induced current was also inhibited 83.0 +/- 8.3% by phorbol 12-myristate 13-acetate (100 nM, n = 6). The inhibition of the cromakalim-induced current by acetylcholine (1 microM) and phorbol 12-myristate 13-acetate (100 nM) was reduced to 27.3 +/- 3.1% (n = 10) and 32.8 +/- 7.8% (n = 3), respectively, in the presence of staurosporine (100 microM). We conclude that muscarinic receptor stimulation inhibits KATP channel activity through the activation of protein kinase C. These findings suggest that KATP channels may play a role in the regulation of membrane potential and contractility in airway smooth muscle.

Frequency modulation of acetylcholine-induced oscillations in Ca++ and Ca(++)-activated Cl- current by cAMP in tracheal smooth muscle.

The effects of adenosine 3':5'-cyclic monophosphate (cAMP) on acetylcholine (ACh)-induced oscillations in intracellular calcium concentration ([Ca++]i) and Ca(++)-activated Cl- current (ClCa current) were determined in isolated tracheal smooth muscle cells. Whole-cell current was measured in individual smooth muscle cells with patch clamp methodology. At a holding potential of -80 mV, ACh (0.1 microM) elicits base line-separated oscillations in ClCa current which correlate with oscillations in [Ca++]i. The addition of the beta adrenoceptor agonist isoproterenol (ISO) (10 nM to 1 microM) in the continued presence of ACh caused a concentration-dependent decrease in the frequency of the oscillations in ClCa current with significant reductions in oscillation frequency of 21.4 and 81.5% in the presence of 0.01 and 0.1 microM ISO, respectively (P < .05). This effect was mimicked by both forskolin (FSK) (3 microM) and 3-isobutyl-1-methylxanthine (IBMX) (30 microM). ISO and forskolin also inhibited ACh-induced oscillations in [Ca++]i measured by confocal fluorescence microscopy in non-voltage-clamped cells loaded with the Ca(++)-sensitive dye, fluo3. The inhibition of ACh-induced oscillations in ClCa current by ISO was partially reversed by increasing extracellular Ca++. These data are consistent with previous observations that the frequency of ACh-induced oscillations in [Ca++]i and ClCa current is dependent on the concentration of extracellular Ca++ and the influx of Ca++ through a verapamil-sensitive pathway. Moreover, these results lend support to the hypothesis that beta adrenoceptors inhibit the ACh-induced increase in [Ca++]i through a cAMP-dependent mechanism that inhibits Ca++ influx and in independent of changes in membrane potential.

Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes.

In macrophages and certain other cell types, extracellular ATP4- can increase plasma membrane permeability through activation of the P2z purinergic receptor. This permeability change involves the induction of non-selective pores which are permeable to molecules with M(r) < or = 900. Electrophysiological studies indicate that agonist occupation of P2z purinergic receptors can additionally activate cation channels which may be distinct from the non-selective pores. We have observed that mammalian P2z purinergic receptors can be expressed in Xenopus oocytes injected with mRNA from the BAC1.2F5 murine macrophage cell line. Under voltage-clamp analysis, these oocytes exhibit a multiphasic inward current in response to ATP or 3'-O-(4-benzoyl)-benzoyl-ATP (BzATP), a selective agonist for the P2z purinergic receptor. This ATP/BzATP-induced current is characterized by a rapidly activated phase which is followed by a delayed, but steady, increase in conductance. We have used two-electrode voltage-clamp analysis and ion substitution to further characterize these P2z purinergic receptor-induced currents as expressed in mRNA-injected oocytes. N,N-Hexamethylene amiloride (HMA), a potent inhibitor of various exchangers and channels, selectively and reversibly inhibited the delayed component of the BzATP-induced inward current. This delayed HMA-sensitive current can be carried by large organic cations, such as N-methyl-D-glucamine (NMG+) and Tris+, in addition to small inorganic cations including Na+, Li+, and K+. In contrast, the rapidly activated HMA-insensitive current is readily carried by Na+, Li+, and K+, but is poorly carried by NMG+ and Tris+. Additional studies characterized P2z receptor regulation of Ca2+ influx, depolarization, ethidium uptake, and fura-2 loss in native BAC1.2F5 macrophages. Reduced temperature permitted discrimination of two distinct permeability pathways which could be activated by BzATP. At 20 degrees C, BzATP did not significantly increase membrane permeability to NMG+ (M(r) 195), ethidium+ (M(r) 314), or fura-2 (M(r) 831) but did stimulate an ion conducting pathway which could be competitively permeated by Na+ or Ca2+. At 37 degrees C, BzATP treatment increased membrane permeability to NMG+, ethidium+, and fura-2, in addition to Na+ and Ca2+. These data indicate that macrophage P2z purinergic receptors can be differentially coupled to: 1) a rapidly gated cation channel, and 2) a time- and temperature-dependent formation of non-selective pores. These two permeability pathways can be distinguished by their rates of activation, ion selectivities, sensitivities to amiloride analogs, and temperature dependence.

Expression of the pore-forming P2Z purinoreceptor in Xenopus oocytes injected with poly(A)+ RNA from murine macrophages.

Extracellular ATP activates two distinct types of P2 purinoreceptor-mediated signaling pathways in macrophages, 1) the rapid formation of nonselective pores/channels in the plasma membrane and 2) a guanine nucleotide-binding protein-dependent stimulation of phosphotidylinositol-specific phospholipase C, with subsequent mobilization of intracellular Ca2+. Several studies have suggested that the pore-forming, or P2z, purinoreceptor may be involved in the cytolytic effects of ATP on macrophages and other cell types. We have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) and UTP as selective agonists for the P2z purinoreceptor and Ca(2+)-mobilizing nucleotide receptor, respectively, in BAC1.2F5 macrophages. In this paper we demonstrate that BzATP and ATP (but not UTP) activate membrane depolarization in BAC1.2F5 cells and also stimulate appropriate electrophysiological responses, consistent with the expression of the P2z purinoreceptor, in Xenopus oocytes injected with poly(A)+ RNA derived from BAC1.2F5 cells. Micromolar concentrations of BzATP or millimolar concentrations of ATP induced a sustained increase in the membrane holding current in these voltage-clamped oocytes. This response was significantly potentiated in the absence of extracellular divalent cations, consistent with the specificity of the P2z purinoreceptor for tetrabasic nucleotides. The sustained currents induced by BzATP or ATP were distinct from the transient and/or oscillating increases in Ca(2+)-dependent Cl- currents that were stimulated by UTP but not BzATP. UTP-stimulated transient currents and nucleotide-dependent increases in aequorin luminescence in poly(A)+ RNA-injected oocytes were independent of extracellular Ca2+ and were correlated with the mobilization of intracellular Ca2+ stores. Sucrose fractionation of the poly(A)+ RNA from BAC1.2F5 cells resulted in the enrichment of mRNA species encoding the components of the P2z purinoreceptor, as well as the Ca(2+)-mobilizing nucleotide receptor, in fractions containing 2.5-4.0-kilobase species.

Chronic treatment with P2-purinergic receptor agonists induces phenotypic modulation of the HL-60 and U937 human myelogenous leukemia cell lines.

In previous studies we have demonstrated that extracellular ATP (and UTP), acting through P2-purinergic receptors, can stimulate the inositol phospholipid signaling system in neutrophils and monocytes, as well as in neutrophil/monocyte progenitor cells. In this study we have examined the ability of extracellular nucleotides to modulate the phenotype of myelomonocytic progenitor cells. As model systems, we utilized the established HL-60 promyelocytic and U937 promonocytic human cell lines which were cultured in the continuous presence of nucleotides known to be potent agonists for P2-purinergic receptors. When cultured for 5 days with ATP gamma S (a phosphatase resistant analog of ATP) plus 10% fetal bovine serum, both HL-60 cells and U937 cells expressed several (but not all) phenotypic characteristics of differentiated phagocytes. In HL-60 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides, (2) a reduction in cell size with a decreased nuclear/cytoplasmic ratio, (3) a sharply reduced rate of proliferation, (4) a reduction in the percentage of cells expressing surface transferrin receptors, and (5) an increase in the percentage of cells expressing the type 1 complement receptor (CR1). In U937 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides and platelet activating factor, (2) a reduced rate of proliferation, (3) a reduction in the percentage of cells expressing surface transferrin receptors, and (4) increases in the percentage of cells expressing both type 1 (CR1) and type 3 (CR3) complement receptors. During the first 12-24 hr after exposure to ATP gamma S, HL-60 cells showed no obvious changes in morphology, viability, or the levels of beta-actin mRNA, but did show (1) a 4-fold increase in chemotactic peptide-induced Ca2+ mobilization, and (2) a greater than 90% decrease in c-myc mRNA levels. Significantly, when HL-60 cells were treated under serum-free conditions, the ability of ATP to enhance expression of functional FMLP receptors could be dissociated from the inhibitory effects of adenine nucleotides on cell proliferation observed in serum containing media. Moreover, treatment of serum-free HL-60 cultures with UTP, another P2-purinergic receptor agonist, also resulted in enhanced expression of functional FMLP receptors.

Plasma and white adipose tissue lipid composition in marmots.

White adipose tissue biopsies and plasma samples were obtained from hibernating yellow-bellied marmots (Marmota flaviventris) maintained in the laboratory. In addition, biopsies and plasma samples were obtained from normothermic animals in the field and laboratory. Measurement of plasma free fatty acid (FA) levels indicated that winter laboratory animals exhibited increased lipolysis. Additionally, analysis of white adipose tissue triacylglycerol revealed that the FA composition of the storage fat in animals maintained on the standard laboratory diet is remarkably simple and uniform between different adipose depots in the same animal. Three FAs (palmitic, oleic, and linoleic acids) made up greater than 95% of the total. Triene (alpha-linolenate) was found in newly captured animals, but the percentage of this FA decreased rapidly when the animals were maintained on the standard laboratory diet. Throughout the hibernation season (October to April), white adipose tissue-saturated FA percentage decreased, monoene percentage remained constant, and diene percentage increased. Analysis of plasma FA composition suggested that these animals tended to metabolize saturated FAs from stored lipid during hibernation and that dienes were mobilized briefly after the last arousal from hibernation in spring. From these observations, we hypothesize that marmots preferentially metabolize saturated fats during the hibernation period and that essential FAs of the omega 6 series tend to be metabolized more slowly than other FAs. These characteristics suggest that marmots are a valuable animal model in which to study lipid metabolism.