A practical approach for the detection of DNA nanostructures in single live human cells by fluorescence microscopy.

In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, cell cycle stage, make the cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. In this paper, we describe the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with epifluorescence microscopy. We report that HeLa cells internalize these nanostructures spontaneously with a higher efficiency with respect to single-stranded or double-stranded oligonucleotides. Our findings suggest that DNA tetrahedra could serve as a platform for the realization of a series of multifunctional intracellular biosensors for the analysis of single live cells.

Serotonin uptake and metabolism by cultured guinea pig airway smooth muscle cells.

Serotonin (5-hydroxytryptamine, 5-HT) is synthesized and released in the airways by pulmonary neuroendocrine cells located in the vicinity of airway smooth muscle (ASM). The aim of this study was to determine whether ASM cells contribute to the inactivation of serotonin, and investigate the role of the serotonin transporter (SERT) and monoamine oxidase (MAO) in this process. Cultured guinea pig tracheal smooth muscle cells, maintained in culture medium containing serotonin for 1-4 days, induced a decrease in 5-HT and increase in 5-HIAA in the culture medium. Changes in indole concentrations were prevented by fluvoxamine and iproniazid. Na+-sensitive [3H]-serotonin uptake into cultured ASM cells was time- and concentration-dependent (Km, 561 nM; Vmax, 1.06 pmol/mg protein/min), and inhibited by clomipramine (IC50, 13.7 nM), fluvoxamine (IC50, 0.16 microM) and fluoxetine (IC50, 0.32 microM). Western blot analysis with an anti-SERT antibody revealed a single 115 kDa immunoreactive band in ASM cell lysates. The results of this study suggest that ASM contributes to the uptake and metabolism of serotonin via SERT and MAO, respectively, and may therefore play a role in the inactivation of endogenous serotonin generated within the airway wall.

Bradykinin increases Na(+)-K(+) pump activity in cultured guinea-pig tracheal smooth muscle cells.

1. The effect of bradykinin on the Na+-K+ pump of airway smooth muscle was investigated by measuring ouabain-sensitive (86)Rb(+) uptake in cultured guinea-pig tracheal smooth muscle cells. 2. Bradykinin induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake, with an EC(50) of 3 nM (pD(2) = 8.50+/-0.10). Stimulation was not affected by indomethacin (1 microM) suggesting that it is not mediated by cycloxygenase products of arachidonic acid. 3. The B(1) receptor agonists Lys-des-Arg(9)-bradykinin and des-Arg(9)-bradykinin had no effect on ouabain-sensitive (86)Rb(+) uptake. In contrast, the B(1) and B(2) receptor agonist Lys-bradykinin induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake with an EC(50) of 6 nM (pD(2) = 8.21 +/- 0.20). 4. The B(1) receptor antagonist des-Arg(10)-HOE 140 (1 microM) had no effect on bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake. The B(2) receptor antagonists HOE 140 and WIN 64338 antagonized bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake with pK(B) values (-log M) of 8.20 +/- 0.08 and 8.11 +/- 0.20 respectively. 5. Reducing extracellular Na+ from 146 mM to 11 mM caused a 53.5% decrease in basal ouabain-sensitive (86)Rb+ uptake and abolished bradykinin-induced uptake. Two inhibitors of the Na(+)-H(+) exchanger, methylisobutyl-amiloride (MIA; 1 - 100 microM) and ethylisopropyl-amiloride (EIPA; 0.1 - 10 microM), inhibited bradykinin-stimulated ouabain-sensitive (86)Rb(+) uptake without affecting basal uptake. 6. These results suggest that bradykinin increases Na+-K+ pump activity of guinea-pig tracheal smooth muscle via stimulation of B(2) receptors and activation of the Na+-H+ exchanger.

Inhibition of vascular smooth muscle tone by the H+, K+-ATPase inhibitor SCH 28080.

Vascular smooth muscle is thought to possess an H+-K+ ATPase that contributes to the regulation of intracellular K+ concentration and pH. We have examined the effect of the H+, K+-ATPase inhibitor SCH 28080 on vascular smooth muscle tone in guinea-pig and human isolated arteries, and on 86Rb+ uptake in cultured guinea-pig aortic smooth muscle cells. SCH 28080 (0.1-300 microM) produced relaxation of isolated guinea-pig aorta, guinea-pig pulmonary artery and human pulmonary artery. Relaxation occurred in tissues pre-contracted with phenylephrine, histamine or the thromboxane mimetic U44069. Relaxation. was reversible, and was not affected by tetrodotoxin, indomethacin, nordihydroguiaretic acid (NDGA), 1-aminobenzotriazole (1-ABT), N(G)-nitro-L-arginine methyl ester (L-NAME), removal of the endothelium or removal of extracellular K+. SCH 28080 had no effect on 86Rb+ uptake in cultured guinea-pig aortic smooth muscle cells. In conclusion, SCH 28080 relaxes vascular smooth muscle at concentrations known to inhibit the H+-K+ ATPase. The persistence of relaxation in a K+-free medium and the failure of SCH 28080 to inhibit 86Rb+ uptake suggest that relaxation may be unrelated to H+, K+-ATPase inhibition, and indicate that this agent may not be considered as a selective H+, K+-ATPase inhibitor in vascular preparations.

Stimulation of the Na(+)-K(+) pump in cultured guinea pig airway smooth muscle cells by serotonin.

The effect of 5-hydroxytryptamine (5-HT) or serotonin on Na(+)-K(+) pump activity of airway smooth muscle was investigated by measuring (86)Rb(+) uptake in cultured guinea pig tracheal smooth muscle cells. (86)Rb(+) uptake consisted of three distinct components, one sensitive to ouabain, one to bumetanide, and one insensitive to either inhibitor. 5-HT induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake (EC(50) = 21 nM) but had no effect on bumetanide-sensitive uptake, suggesting that it stimulates the Na(+)-K(+) pump but not the Na(+)-K(+)-Cl(-) cotransporter. Ouabain-sensitive uptake also was stimulated by the 5-HT(2A/2C) agonists 2,5-dimethoxy-4-iodoamphetamine and alpha-methyl-5-HT, but not by the 5-HT(1) agonist 5-carboxamidotryptamine, the 5-HT(1A/1B/2C) agonist 1-(3-chlorophenyl)piperazine, or the 5-HT(3) agonist 1-(3-chlorophenyl)biguanide. 5-HT-stimulated (86)Rb(+) uptake was inhibited by the 5-HT(2A) antagonists ketanserin and spiperone, but not by the 5-HT(1A) antagonist NAN 190 or the 5-HT(3) antagonist Y25310. 5-HT-stimulated (86)Rb(+) uptake was inhibited by reducing extracellular Na(+) concentration and by the Na(+)-H(+) exchange inhibitors dimethylamiloride and 5-(N-methyl-N-isobutyl)-amiloride. These observations suggest that 5-HT stimulates the Na(+)-K(+) pump of airway smooth muscle via 5-HT(2A) receptors by a mechanism dependent on Na(+) influx, possibly through the Na(+)-H(+) exchanger. Because stimulation of the Na(+)-K(+) pump produces hyperpolarization, this may represent a negative-feedback mechanism that opposes contraction in response to 5-HT.

Binding of guanine nucleotides to bronchial membranes: effect of maturation.

Guanosine 5-[y-thio]triphosphate ([35S]GTP gamma S) binding to guinea pig bronchial membranes from immature and mature guinea pigs was rapid (Kon: 3.8 x 10(5) mol-1 min-1), saturable (Bmax: 160 pmoles/mg protein) and of high affinity (Kd: 0.6 microM). [35S]GTP gamma S rapidly dissociated in the absence of magnesium (Koff: 0.06 min-1), but 50 mM magnesium inhibited the dissociation. Maturation did not alter the affinity of the ligand, but Bmax (pmoles/mg DNA) was greater in preparations from mature animals (929 +/- 16 vs. 620 +/- 64). [35S]GTP gamma S was displaced by guanine nucleotides with a rank order of potency of GDP beta S = Gpp(NH)p > GDP > GTP, but not by ATP. We conclude that [35S]GTP gamma S is a specific and useful method to quantitate bronchial membrane-bound GTP-binding proteins. The technique shows that there is a significant increase in the cellular content of G-proteins during maturation.

Expression of HMGI-C and HMGI(Y) in ordinary lipoma and atypical lipomatous tumors: immunohistochemical reactivity correlates with karyotypic alterations.

The high mobility group proteins (HMGs) are a class of low molecular weight, nonhistone, nuclear proteins that bind DNA and function as transcription cofactors. This class includes the HMGI family members HMGI-C and HMGI(Y). Both are not significantly expressed in differentiated adult tissues, including fat, but their expression is induced in proliferating and transformed cells. Their involvement in the development of lipomatous tumors has been recently demonstrated for HMGI-C, which is encoded by a gene located at 12q15, the chromosomal segment often rearranged in ordinary lipomas. The same chromosomal segment is consistently amplified in the ring and giant marker chromosomes of atypical lipomatous tumors (ALTs), a term used to designate tumors previously labeled as well differentiated liposarcomas or atypical lipomas. The involvement of HMGI(Y) is strongly suspected as the gene coding for HMGI(Y) is located at 6p21, a chromosomal segment rearranged in a subset of ordinary lipomas. HMGI-C or HMGI(Y) protein expression was analyzed immunohistochemically in a group of 39 well differentiated adipose neoplasms (19 lipomas and 20 ALTs) of known karyotype using polyclonal antibodies raised against a recombinant protein (HMGI-C) and against a synthetic peptide (HMGI(Y)). The results of this study demonstrate that HMGI proteins are commonly expressed in well differentiated adipose neoplasms. Seventeen of twenty ALTS (85.0%), all of which had ring or giant marker chromosomes with amplification of 12q13-15, strongly expressed HMGI-C. HMGI-C expression was detected in 7 of 11 ordinary lipomas (63.6%) with alterations at 12q14-15 and in one case with an abnormal karyotype that included double minute chromosomes. HMGI-C immunoreactivity correlates with 12q13-15 chromosomal alterations (P = 0.001). HMGI(Y) reactivity was demonstrated in only two ordinary lipomas: one with 6p21 rearrangement and one with normal karyotype. No significant HMGI(Y) expression was found in the ALT group. The finding of aberrant expression of HMGI proteins in well differentiated adipose neoplasms in association with 12q13-15 and 6p21 chromosomal changes supports the proposed pathogenetic role of this group of proteins in the development of adipose tissue tumors.

Maturational changes in Na(+)-K+ pump activity in guinea pig airway smooth muscle.

The effect of maturation on the Na(+)-K+ pump of airway smooth muscle (ASM) was studied. Na(+)-K+ pump activity of tracheal smooth muscle from immature (1- to 2-week-old) and mature (10- to 12-week-old) guinea pigs was measured as the ouabain-sensitive component of the resting membrane potential and as ouabain-sensitive 86Rb+ uptake (using 86Rb+ as a marker for K+). Maturation resulted in decreases in both the contribution of the Na(+)-K+ pump of the resting membrane potential and in ouabain-sensitive 86Rb+ uptake. 86Rb+ efflux from tracheal smooth muscle was similar in tissues from immature and mature guinea pigs, suggesting that maturation has no effect on K+ (or at least 86Rb+) permeability. Na+ and K+ contents of tracheal smooth muscle were estimated from the uptakes of 24Na+ and 86Rb+ at equilibrium. Maturation resulted in a decrease in Na+ content but had no effect on K+ content. Since intracellular Na+ is one of the principal determinants of Na(+)-K+ pump activity, these results suggest that maturation results in a decrease in Na(+)-K+ pump activity in ASM, which may be due to a decrease in Na+ content.

H+-K+ ATPase inhibitors cause relaxation of guinea pig and human airway smooth muscle in vitro.

The effect of H+-K+ ATPase inhibitors on airway smooth muscle tone was investigated in vitro. Four H+-K+ ATPase inhibitors, SCH 28080 (2-methyl-8-(phenylmethoxy)-imidazo[1,2-a] pyridine-3-acetonitrile), SK&F 96067 (3-butyryl-4-(2-methylphenylamino)-8-methoxy-quinoline), omaprezole (5-methoxy-2-(((4-methoxy-3,5-dimethyl-2-pyridinyl)-methyl)-sulfinyl)-1H -benzimidazole) and NC-1300-B (2-(2-dimethylaminobenzylsulfiny)-5-methoxybenzimidazole), induced concentration-dependent relaxation of guinea pig trachea with spontaneous tone, with IC50 values of 5.9, 7.1, 155 and 79 microM, respectively, SCH 28080 and omeprazole also relaxed airways precontracted with carbachol or histamine in the presence of indomethacin. Relaxation was similar in intact and epithelium-denuded tracheal preparations, suggesting that the airway epithelium neither mediates or modulates relaxation induced by H+-K+ ATPase inhibitors. SCH 28080-induced relaxation was not influenced by tetrodotoxin, suggesting that it is not neurogenically mediated. Bafilomycin A1 and concanamycin A had no effect on guinea pig tracheal spontaneous tone, suggesting that relaxation induced by H+-K+ ATPase inhibitors is not due to an interaction with a vacuolar H+ ATPase. SCH 28080 also induced concentration-dependent relaxation of human bronchi precontracted with histamine. These results demonstrate that several structurally and/or mechanistically distinct H+-K+ ATPase inhibitors cause relaxation of airway smooth muscle in vitro, and suggest that a H+-K+ ATPase or similar pathway may play a role in the maintenance of airway smooth muscle tone.

Evidence of Na-K-Cl cotransport in airway smooth muscle.

The presence of the Na-K-Cl cotransporter in airway smooth muscle was investigated by measuring 86Rb+ (as a marker for K+) and 36Cl- fluxes in the guinea pig trachealis. K+ uptake consisted of 1) a bumetanide- and furosemide-sensitive component, 2) a ouabain-sensitive component, and 3) a bumetanide- and ouabain-insensitive component. Bumetanide and furosemide inhibited K+ uptake with IC50s of 0.18 and 5.6 microM, respectively. Bumetanide-sensitive K+ uptake was reduced by the isosmotic replacement of extracellular Na+ and Cl- with choline and I-, respectively. Bumetanide caused a reduction in Cl- uptake, and the ratio of bumetanide-sensitive K+ to Cl-uptake was approximately 1:1.75. Bumetanide caused a decrease in 86Rb+ efflux, suggesting that the Na-K-Cl cotransporter mediates both K+ influx and efflux in airway smooth muscle. Ouabain caused an increase in bumetanide-sensitive 86Rb+ efflux from the guinea pig trachealis, which was prevented by exposure to a low-Na+ medium, suggesting that Na-K pump inhibition stimulates outward Na-K-Cl cotransport as a result of an increase in intracellular Na+ content.

Stimulation of GTP hydrolysis in guinea pig bronchial membranes by mastoparan.

Guanine nucleotide-binding proteins, or G proteins, play an important role in transmitting information from membrane receptors to intracellular effector systems. Activation of G proteins results in the hydrolysis of GTP, and the measurement of GTPase activity represents a means by which the role of G proteins in signal transduction can be investigated. GTPase activity of guinea pig bronchial membranes was measured as the liberation of 32Pi from [gamma-32P]GTP. GTPase activity was divided into two components, one possessing a high affinity and the other a low affinity for GTP. The contribution of high- and low-affinity GTPase to total hydrolysis was dependent on Mg2+. In the presence of submicromolar Mg2+, high-affinity GTPase represented 65-80% of all activity, whereas in the presence of > or = 26 microM Mg2+, all detectable hydrolysis was due to the low-affinity GTPase. High-affinity GTPase was stimulated by Mg2+ in the 0.15-1.1 microM range (2.5-fold maximal stimulation, apparent Km for Mg2+ 0.31 microM). Mastoparan (1-100 microM) caused a concentration-dependent stimulation of high-affinity (but not low-affinity) GTPase (71 +/- 13% maximal stimulation, EC50 0.38 microM), suggesting that high-affinity GTPase may be due to a G protein. Carbachol (10 microM) and fenoterol (10 microM) had no effect on high-affinity GTP hydrolysis, suggesting that under the conditions described, GTPase activity of bronchial membranes is not activated by muscarinic or beta-adrenergic receptors, respectively.

Potentiation of nonadrenergic neural relaxation in guinea pig airways by a cyclic cAMP phosphodiesterase inhibitor.

We have studied the effect of phosphodiesterase inhibitors on relaxation of guinea pig tracheal smooth muscle in an attempt to elucidate the role of cyclic nucleotides in relaxation to stimulation of inhibitory nonadrenergic noncholinergic (i-NANC) nerves. SK&F 94120 (1-10 microM) potentiated relaxation induced by isoproterenol, vasoactive intestinal peptide (VIP) and electrical field stimulation (EFS) in the presence of atropine and propranolol but had no effect on relaxation induced by sodium nitroprusside. Zaprinast (3-30 microM) potentiated relaxation induced by sodium nitroprusside but not by isoproterenol or VIP. A small potentiation of relaxation to EFS was induced by 30 microM zaprinast but not by lower concentrations. Tetrodotoxin attenuated relaxations induced by EFS suggesting that they are at least partly neurogenic in origin. SK&F 94120 and zaprinast had no effect of tetrodotoxin-resistant relaxation to EFS. The guanylate cyclase inhibitor had no effect on EFS-induced relaxation. These findings suggest that cyclic AMP may mediate relaxation of guinea pig tracheal smooth muscle in response to stimulation of i-NANC nerves, and are in agreement with the view that VIP may be the neurotransmitter released by i-NANC nerves in this tissue.

Epithelial modulation of non-adrenergic, non-cholinergic and vasoactive intestinal peptide-induced responses: role of neutral endopeptidase.

Relaxation of guinea-pig trachea was induced by vasoactive intestinal peptide (VIP) or electrical field stimulation. Mechanical removal of airway epithelium potentiated responses to VIP and attenuated inhibitory non-adrenergic, non-cholinergic (i-NANC) responses to low stimulation frequencies. Phosphoramidon potentiated responses to VIP in intact but not de-epithelialised preparations, and had no effect on i-NANC responses. These findings suggest that neutral endopeptidase localised to the epithelium may modulate relaxation of guinea-pig trachea induced by VIP but not i-NANC nerve-stimulation.

Effect of hydrogen peroxide on guinea-pig tracheal smooth muscle in vitro: role of cyclo-oxygenase and airway epithelium.

1. Hydrogen peroxide (H2O2) (0.1 microM-3 mM) induced variable contractions of guinea-pig isolated trachea which were attenuated by catalase (100 u ml-1) and mannitol (15 mM) suggesting that contractions were induced by H2O2 and/or the hydroxyl anion. 2. Epithelial removal potentiated contractile responses of tracheal preparations to H2O2 with a leftward shift of the concentration-response curve and an increase in the maximal response. 3. Indomethacin (3 microM) inhibited contractions to H2O2 of intact preparations and preparations without epithelium suggesting that contractions may be mediated by cyclo-oxygenase products. Intact preparations (but not preparations without epithelium) contracted in response to high concentrations (greater than 0.1 mM) of H2O2 in the presence of indomethacin suggesting that other excitatory factor(s) released by the epithelium may induce contraction. 4. Preincubation of intact tracheal preparations with H2O2 (1 mM) for 1 h had no effect on responses to histamine or isoprenaline. 5. These results suggest that hydrogen peroxide generated during the inflammatory process may play a role in bronchoconstriction.

Influence of epithelium on guinea pig airway responses to tachykinins: role of endopeptidase and cyclooxygenase.

We have studied the effect of epithelium removal on the contractile responses to exogenous tachykinins and to endogenous tachykinins released by capsaicin in guinea pig trachea. We also studied the effects of inhibition of endopeptidase (by phosphoramidon, 10 microM, and thiorphan, 100 microM), and of inhibition of cyclooxygenase (by indomethacin, 5 microM) on these responses. The order of potency of exogenous tachykinins was neurokinin A (NKA) greater than neurokinin B (NKB) greater than substance P (SP). Epithelium removal enhanced the sensitivity and magnitude of the contractile response to SP, and to a lesser extent NKA and NKB. Capsaicin induced only a weak contractile response in guinea pig trachea. Phosphoramidon and thiorphan increased the sensitivity to SP, but had no effect on acetylcholine responses. The leftwardshift due to epithelium removal was reduced, but not abolished, by phosphoramidon and thiorphan. NKA- and NKB-induced contractions were also enhanced significantly by phosphoramidon. The effect of epithelium removal was abolished for NKA, but not for NKB. Phosphoramidon also increased significantly the contraction to capsaicin in the presence of epithelium, without altering the response obtained in the absence of epithelium. Indomethacin potentiated the sensitivity and maximal contractile response to all the tachykinins with the greatest effect on SP responses, and to capsaicin. The combination of indomethacin with phosphoramidon or thiorphan abolished the effect of epithelium removal for all the tachykinins. We conclude that the effects of exogenous and endogenous tachykinins are enhanced by removal of epithelium and by inhibition of metalloendopeptidase and cyclooxygenase, suggesting that tachykinins may be degraded by epithelial enzymes, and may release relaxant prostanoids in airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cholinergic neurotransmission in human airways by beta 2-adrenoceptors.

The purpose of the study was to determine whether catecholamines modulate cholinergic neurotransmission in isolated human airway smooth muscle. Bronchial rings were suspended in organ baths for isometric measurement of tension, and contractions were induced by either electrical field stimulation (EFS) or exogenous acetylcholine (ACh). Isoproterenol, epinephrine, and norepinephrine in that order of potency produced concentration-dependent inhibition of comparable responses to EFS and ACh. However a potency difference of 100-fold for isoproterenol (IC50 = 4.80 X 10(-8) M for EFS and 3.70 X 10(-6) M for ACh) and 10-fold for both epinephrine and norepinephrine was observed for inhibition of responses to EFS compared with responses to ACh. The inhibitory effects of isoproterenol on responses to EFS were prevented by propranolol and ICI 118551 (a beta 2-antagonist) but not by betaxolol (a beta 1-antagonist). Tyramine had no effect on contractions elicited by EFS. These experiments demonstrate that beta-agonists inhibit cholinergic nerve-induced contractions of human bronchi more potently than contractions induced by exogenous ACh, suggesting modulation of cholinergic neurotransmission by prejunctional beta 2-receptors.

The effect of platelet-activating factor on histamine and muscarinic receptor function in guinea pig airways.

Platelet-activating factor (PAF) administered i.v. or by aerosol to guinea pigs elicited an increase in airway responsiveness to both acetylcholine and histamine when compared with guinea pigs exposed to the vehicle bovine serum albumin (BSA). After i.v. PAF, the ED100 for acetylcholine and histamine was 5.4 and 3.4 micrograms/kg, respectively, in comparison with 17 and 6 micrograms/kg, respectively, before PAF, representing approximately a 3- and 2-fold increase in responsiveness, respectively. After aerosol PAF (500 micrograms), the ED100 for acetylcholine and histamine was 13 and 7 micrograms/kg, respectively, whereas after aerosolized BSA, the ED100 for acetylcholine and histamine was 23 and 12 micrograms/kg, respectively, representing approximately a 2-fold increase in responsiveness. However, airway smooth muscle obtained from these PAF- or BSA-treated animals did not exhibit any differences in contractile response to histamine or acetylcholine in vitro. Likewise, there were not significant differences in the binding affinity or receptor density between PAF- and BSA-treated tissues with [3H]quinuclidinylbenzilate or [3H]pyrilamine binding, which were used to identify muscarinic and H1-histamine receptors, respectively. Furthermore, histamine and carbachol-induced phosphoinositide hydrolysis was similar in PAF- and BSA-treated tracheal smooth muscle preparations. Thus, PAF induces airway hyperresponsiveness in the guinea pig that is not related to changes in airway smooth muscle or to changes in muscarinic and histamine (H1) receptor density or function.

Tachykinin-induced phosphoinositide breakdown in airway smooth muscle and epithelium: relationship to contraction.

We have studied the contractile response and phosphoinositide hydrolysis induced by substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and Alp-Phe-Phe(R)-Gly[ANC-2]-Leu-Met-NH2 (L 363851), a selective NK2-receptor agonist, in guinea pig tracheal smooth muscle. The four tachykinins elicited a concentration-dependent contraction in tracheal smooth muscle devoid of epithelium, with the following order of potency: NKA greater than L 363851 greater than NKB greater than SP, (EC50 1.0 x 10(-9) M, 3.2 x 10(-9) M, 7.5 x 10(-9) M and 1.2 x 10(-7) M, respectively), which suggests that NK2 receptors predominate in airway smooth muscle. In the presence of epithelium, the sensitivity of airway smooth muscle to tachykinins was decreased, and the concentration response curves to tachykinins were shifted rightward by 30-fold for SP, 9-fold for NKA, and 5-fold for NKB. The concentration response curve to L 363851 was not significantly shifted in the presence of epithelium. This suggests that epithelium may release a relaxant factor in response to tachykinins via an NK1 receptor. In airway smooth muscle, we found that tachykinins elicited phosphoinositide breakdown with an order of potency similar to that for contractile response (EC50 2.2 x 10(-5) M, 3.6 x 10(-5) M, 4.4 x 10(-5) M, and 5.9 x 10(-5) M). In epithelium, SP alone elicited a significant phosphoinositide breakdown, suggesting that epithelial receptors to tachykinins may be of the NK1 subtype. Since it is established that phosphoinositide derivatives can elicit mobilization of intracellular calcium, our results suggest that phosphoinositide breakdown is the coupling mechanism for tachykinin-induced contraction of airway smooth muscle.

The effect of platelet activating factor on pulmonary beta-adrenoceptors.

An intravenous infusion of platelet activating factor (Paf) in the guinea-pig elicits an increase in bronchial responsiveness to the spasmogens, histamine and bombesin. Airways obstruction induced by bombesin in Paf-treated animals is poorly reversed by isoprenaline compared to comparable airways obstruction induced by bombesin in vehicle-treated animals. Isoprenaline induced a comparable dose-related relaxation in vitro of tracheal smooth muscle isolated from Paf- and vehicle-treated animals. No change in beta-adrenoceptor numbers or binding affinity was observed in lungs removed from Paf-treated animals in comparison with those from vehicle-treated animals, or after direct incubation with Paf in vitro. The reduced bronchodilator responsiveness to isoprenaline in Paf-treated animals is not related to changes in pulmonary beta-adrenoceptor function. These results suggest that non-spasmogenic elements may contribute to airways obstruction induced in hyper-responsive animals.

Prolonged apnea and cardiac arrhythmias in infants discharged from neonatal intensive care units: failure to predict an increased risk for sudden infant death syndrome.

Twenty-four hour recordings of respiratory wave form and ECG were made on low-birth-weight and/or premature infants within one week of discharge from eight neonatal intensive care units. Eight infants (0.7%) had episodes of apnea greater than 30 seconds in duration, all of which were accompanied by bradycardia less than 100 beats per minute; 25 infants (2.3%) had a total of 36 apneic episodes between 20 and 30 seconds in duration, 29 of which were accompanied by bradycardia less than or equal to 100 beats per minute; and 19 infants (1.7%) had episodes of bradycardia less than or equal to 50 beats per minute without prolonged apnea (as shown by a lack of breathing movement). Five infants had ventricular premature beats (including one with ventricular tachycardia). Eleven infants had supraventricular premature beats (including two with supraventricular tachycardia and one with preexcitation). Four infants had both supraventricular and ventricular premature beats. Two infants had preexcitation. Eleven infants who underwent 24-hour recordings died. Five infants were victims of sudden infant death syndrome. One infant death was sudden and unexpected and was attributed to bronchopneumonia. Two deaths were associated with congenital heart disease and three were associated with major cerebral disorders. None of the six babies who died suddenly and unexpectedly had apnea greater than or equal to 20 seconds, bradycardia less than or equal to 50 beats per minute, or cardiac arrhythmias on their 24-hour recordings.