Robert R. Lorenz

Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig.

1 The possible interactions between prostacyclin and endothelium‐derived relaxing factor were examined, in isolated coronary arteries of the pig treated with indomethacin (10−5 m). 2 In organ chamber experiments, prostacyclin caused relaxations, which were potentiated in the presence of the endothelium; the potentiation was abolished by oxyhaemoglobin. 3 In bioassay experiments, prostacyclin caused minimal relaxations of bioassay rings without endothelium; these relaxations were potentiated when the bioassay ring was exposed to basally‐released endothelium‐derived relaxing factor (interaction between prostacyclin and basal endothelium‐derived relaxing factor) and further augmented when the endothelial cells were exposed to the prostanoid (stimulated release of endothelium‐derived relaxing factor). The endothelium‐dependent, but not the direct effects of prostacyclin were augmented by superoxide dismutase plus catalase and abolished by oxyhaemoglobin. 4 Forskolin, a direct activator of adenylate cyclase, caused relaxations of rings without endothelium, which were augmented by the presence of the endothelium. 5 The relaxations induced by prostacyclin or forskolin also had an endothelium‐dependent component in basilar and femoral arteries and in jugular veins of the pig. 6 The endothelium‐dependent actions of prostacyclin probably reflect activation of adenylate cyclase.

Release of different relaxing factors by cultured porcine endothelial cells.

Experiments were performed to determine the effect of ouabain on the release of relaxing factor(s) from cultured endothelial cells, and its action on the effect of the relaxing factor(s) on arterial smooth muscle. A column of porcine aortic endothelial cells grown on microcarrier beads in suspension culture was perfused with modified Krebs-Ringer bicarbonate solution. The release of relaxing factor(s) by the endothelial cells was detected under bioassay conditions by measuring the relaxing activity of the perfusate overflowing a ring of canine coronary artery (without endothelium) contracted with prostaglandin F2α. Incubation of the endothelial cells with ouabain did not affect the relaxation of the bioassay ring under basal conditions or upon stimulation of the endothelial cells with ADP but impaired the relaxation Induced by bradykinin or the calcium ionophore A23187. Incubation of the bioassay ring with ouabain reduced the relaxation under basal conditions as well as the relaxation induced by ADP but did not affect the relaxation observed upon stimulation with bradykinin and A23187 and the endothelium- independent relaxations induced by nitric oxide. These experiments suggest that cultured porcine aortic endothelial cells release two endothelium-derived relaxing factors; one is released under basal conditions and upon stimulation with adenosine diphosphate and the other (which presumably is nitric oxide) upon stimulation with bradykinin and the calcium ionophore A23187.

F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscle in dogs

1 Dynamic actin reorganization involving actin polymerization and depolymerization may play an important functional role in smooth muscle. 2 This study tested the hypothesis that F‐actin stabilization by phalloidin increases tension cost (i.e. ATP hydrolysis rate per unit of isometric force) during Ca2+‐induced activation of Triton X‐100‐permeabilized canine tracheal smooth muscle. 3 Adenosine 5′‐triphosphate (ATP) hydrolysis rate was quantified using an enzyme‐coupled NADH fluorometric technique, regulatory myosin light chain (rMLC) phosphorylation was measured by Western blot analysis, and maximum unloaded shortening velocity (Vmax) was estimated by interpolation of the force‐velocity relationship to zero load during isotonic loading. 4 Maximal activation with 10 μm free Ca2+ induced sustained increases in isometric force, stiffness, and rMLC phosphorylation. However, the increase in ATP hydrolysis rate initially reached peak values, but then declined to steady‐state levels above that of the unstimulated muscle. Thus, tension cost decreased throughout steady‐state isometric force. 5 Following incubation of permeabilized strips with 50 μm phalloidin for 1 h, the increases in isometric force and stiffness were not sustained despite a sustained increase in rMLC phosphorylation. Also, after an initial decline, tension cost increased throughout activation. Phalloidin had no effect on Vmax during steady‐state isometric force or on rMLC phosphorylation. 6 These findings suggest that dynamic reorganization of actin is necessary for optimal energy utilization during contraction of permeabilized airway smooth muscle.

Response of human umbilical artery to changes in transmural pressure

Human umbilical artery was perfused with Krebs-Ringer bicarbonate-glucose medium at 37 C and pH 7.4. An increase in transmural pressure elicited a vasoconstriction. The artery wall contained no sig...

The effects of acetylstrophanthidin and ouabain on the sympathetic adrenergic neuroeffector junction in canine vascular smooth muscle.

We performed experiments to determine the effects of acetylstrophanthidin (ACS) and ouabain on the adrenergic neuroeffector junction in dog saphenous veins. In quiescent strips incubated with3H-norepinephrine (3H-NE), the drugs caused contraction and a progressive increase in overflow of3H-NE and O-methylated metabolites; 3,4-dihydroxyphenylglycol (DOPEG) decreased. Tissue uptake of3H-NE was partially inhibited. After surgical sympathectomy, both contraction and3H-NE overflow were markedly attenuated. Following chemical sympathectomy with 6-hydroxydopamine, ouabain contractions were 11% of control, whereas the contractions due to exogenous norepinephrine were exaggerated. The initial overflow of3H-NE was unaffected by tetrodotoxin, but the later and larger overflow with prolonged exposure was depressed. The former occurred in the absence of Ca2+, but the latter was Ca2+dependent. Inhibition of the neuronal amine carrier by cocaine or desipramine and blockade of the neuronal a-adrenoceptors with phentolamine or phenoxybenzamine attenuated the release of3H-NE evoked by ACS and ouabain. During electrical stimulation, ACS augmented the overflow of3H-NE. This was attenuated by cocaine, desipramine, and the a-adrenolytic drugs. ACS, like pargyline, augmented the overflow of3H-NE evoked by tyramine and depressed that of DOPEG. These experiments suggest that acetylstrophanthidin and ouabain (1) cause contraction of vascular smooth muscle by displacement of norepinephrine from neuronal stores, (2) reduce neuronal mono-amine oxidase activity, (3) facilitate and may trigger Ca2+-dependent exocytotic release of norepineph-rine, (4) partially inhibit the neuronal amine carrier mechanism but do not interfere with extraneuronal disposition of norepinephrine, and, finally (5) may have unexplained interactions with prejunctional a-adrenoceptors. Circ Res 47: 845-854, 1980

Role of protein kinase C in calcium sensitization during muscarinic stimulation in airway smooth muscle

Muscarinic receptor stimulation increases Ca2+ sensitivity, i.e., the amount of force produced at a constant submaximal cytosolic Ca2+ concentration ([Ca2+]i), in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+ sensitivity is in part mediated by protein kinase C (PKC). With the use of a beta-escin permeabilized canine tracheal smooth muscle (CTSM) preparation, the effect of four putative PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC [PKC peptide-(19-31)], and staurosporine] on Ca2+ sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+ sensitization induced by muscarinic receptor stimulation in the presence of a constant submaximal [Ca2+]i, neither did any of these compounds reverse the increase in Ca2+ sensitivity induced by ACh plus GTP. Administration of a 1,2-diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, did not induce Ca2+ sensitization at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity produced by muscarinic receptor stimulation in permeabilized CTSM.

Calcium Concentration-dependent Mechanisms through which Ketamine Relaxes Canine Airway Smooth Muscle

Background: Ketamine is a potent bronchodilator that, in clinically used concentrations, relaxes airway smooth muscle in part by a direct effect. This study explored the role of calcium concentration (Ca2+) in this relaxation. Methods: Canine trachea smooth muscle strips were loaded with the fluorescent probe fura‐2 and mounted in a spectrophotometric system to measure force and intracellular calcium concentration ([Ca2+]i) simultaneously. Calcium influx was estimated using a manganese quenching technique. Cyclic nucleotides in the airway smooth muscle strips were measured by radioimmunoassay. Results: In smooth muscle strips stimulated with submaximal (0.1 micro Meter) and maximal (10 micro Meter) concentrations of acetylcholine, ketamine caused a concentration‐dependent decrease in force and [Ca2+]i. The sensitivity of the force response to ketamine significantly decreased as the intensity of muscarinic receptor stimulation increased; the median effective concentration for relaxation induced by ketamine was 59 micro Meter and 850 micro Meter for tissue contracted by 0.1 micro Meter or 10 micro Meter acetylcholine, respectively (P < 0.05). In contrast, the sensitivity of the [Ca2+] sub i response did not depend on the intensity of muscarinic receptor stimulation. Ketamine at 1 mM significantly inhibited calcium influx. Ketamine did not significantly increase cyclic nucleotide concentrations. Conclusions: Ketamine‐induced relaxation of canine airway smooth muscle is associated with a decrease in [Ca2+]i and calcium influx, effects that are not mediated by an increase in cyclic nucleotides; and the sensitivity of the force response to ketamine decreases as the level of preexisting muscle tone increases, an effect that is not explained by differential effects on [Ca2+]i.

Prejunctional beta-adrenoceptors in human and canine saphenous veins

Experiments were designed to compare the functional importance of prejunctional beta-adrenoceptors in human and canine blood vessels. Rings of saphenous veins were mounted for isometric tension recording in organ chambers filled with physiological salt solution. Isoproterenol caused concentration-dependent relaxations of comparable magnitude during contractions of canine veins caused by electrical stimulation or norepinephrine. In the human veins, isoproterenol caused smaller relaxations during norepinephrine-induced contractions, and augmented the contractile response to electrical stimulation. Helical strips of veins were incubated with [3H]norepinephrine and mounted for superfusion and isometric tension recording. Isoproterenol augmented the release of intact labeled transmitter during electrical stimulation more in the human than in the canine vein. In veins from both species the effects of isoproterenol were inhibited by propranolol. These experiments demonstrate that the adrenergic nerves of the human saphenous vein have a greater responsiveness to prejunctional beta-adrenergic activation than those of the corresponding canine vessel. As a consequence, in the human vein, beta-adrenergic agonists augment, rather than depress the contractile response to activation of the adrenergic nerve endings.

Venomotor changes caused by halothane acting on the sympathetic nerves.

Experiments were performed to determine whether depression of venomotor responses with halothane results from interference with sympathetic activation or from an effect on venous smooth muscle cells. Changes in isometric tension of isolated canine saphenous-vein strips were recorded. Adrenergic activation was achieved by transmural electrical stimulation, by addition of tyramine, and by addition of norepinephrine. Halothane (0.5 to 3 per cent) did not significantly alter basal tension. It lessened the reaction of the veins to electrical stimulation but not their response to norepinephrine; it increased the response to tyramine. Since the responses to norepinephriae and tyramine were not decreased, halothane appears to act on the nerve terminal to prevent release of neurotransmitter associated with nerve-terminal depolarization. Thus, halothane causes inhibition of electrically induced venoconstriction in cutaneous veins, probably by interfering with the release of norepinephrine from nerve terminals rather than by an inhibitory effect on the smooth-muscle cells.

Interaction between Neuronal Amine Uptake and Prejunctional Alpha-Adrenergic Receptor Activation in Smooth Muscle from Canine Blood Vessels and Spleen

Experiments were performed to determine the conditions in which norepinephrine release from adrenergic nerve terminals in smooth muscle from canine blood vessels and spleen might be inhibited by prejunctional alpha-adrenergic receptor activation. Strips of aorta, mesenteric and splenic arteries, splenic capsule and portal and saphenous veins were labeled with 7-3H-norepinephrine and mounted for superfusion. In the portal vein, an inhibitory effect of prejunctional receptor activation with exogenous norepinephrine (1.2 X 10(-6) M) on transmitter efflux could be demonstrated during electrical stimulation (9 V, 2 Hz) of the nerve terminals. By contrast, in the other tissues, inhibition of transmitter release during electrical stimulation or depolarization of the nerve terminals with K+ (40 mEq/l) could only be demonstrated aftet blockade of the neuronal uptake mechanism. That activation of prejunctional alpha-adrenergic receptors in blood vessels inhibits the exocytotic process is suggested by the failure of exogenous norepinephrine to affect either the basal efflux of 3-H-norepinephrine or the displacement of 3H-norepinephrine by tyramine.