Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils

Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.     

Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor

The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation.     

Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils?

Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated [32P]PIP3 levels decayed toward initial values. However, recovery of [32P]PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of [32P] PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.     

The second epidermal growth factor-like domain of human factor IXa mediates factor IXa binding to platelets and assembly of the factor X activating complex.

Factor IXa binding to the activated platelet surface is required for efficient catalysis of factor X activation. Platelets possess a specific binding site for factor IXa, occupancy of which has been correlated with rates of factor X activation. However, the specific regions of the factor IXa molecule that are critical to this interaction have not yet been fully elucidated. To assess the importance of the second epidermal growth factor (EGF2) domain of factor IXa for platelet binding and catalysis, a chimeric protein (factor IXa(Xegf2)) was created by replacement of the EGF2 domain of factor IX with that of factor X. Competition binding experiments showed 2 different binding sites on activated platelets (approximately 250 each/platelet): (1) a specific factor IXa binding site requiring the intact EGF2 domain; and (2) a shared factor IX/IXa binding site mediated by residues G(4)-Q(11) within the Gla domain. In kinetic studies, the decreased V(max) of factor IXa(Xegf2) activation of factor X on the platelet surface (V(max) 2. 90 +/- 0.37 pM/min) versus normal factor IXa (37.6 +/- 0.15 pM/min) was due to its decreased affinity for the platelet surface (K(d) 64.7 +/- 3.9 nM) versus normal factor IXa (K(d) 1.21 +/- 0.07 nM), resulting in less bound enzyme (functional complex) under experimental conditions. The hypothesis that the binding defects of factor IXa(Xegf2) are the cause of the kinetic perturbations is further supported by the normal k(cat) of bound factor IXa(Xegf2) (1701 min(-)(1)) indicating (1) an intact catalytic site and (2) the normal behavior of bound factor IXa(Xegf2). The EGF2 domain is not a cofactor binding site since the mutant shows a normal rate enhancement upon the addition of cofactor. Thus, the intact EGF2 domain of factor IXa is critical for the formation of the factor X activating complex on the surface of activated platelets.     

Neurotensin elevates hepatic bile acid secretion in chickens by a mechanism requiring an intact enterohepatic circulation

Neurotensin (NT), given intravenously at 10-50 pmol/kg per min to anesthetized female chickens equipped with a bile duct fistula, dose-dependently elevated hepatic bile flow and bile acid output but only when the enterohepatic circulation was maintained by returning the bile to the intestinal lumen. Infusion of NT at 10 and 50 pmol/kg per min increased the average hepatic bile acid output over a 30-min period to 138 +/- 11 and 188 +/- 13% of control, respectively. During infusion of NT, plasma levels of immunoreactive NT (iNT) increased in time from the basal level (14 +/- 1.3 pM) to reach steady state at 30 min. There was a near linear relationship between the dose of NT infused and the increment in plasma iNT. In addition, infusion of NT at 40 pmol/kg min gave a plasma level of iNT (approximately/= 88 pM) which was within the range of those observed during duodenal perfusion with lipid (54-300 pM) and near to that measured in hepatic portal blood from fed animals (52 +/- 5 pM). Perfusion of duodenum with lipid released endogenous NT and increased the rate of hepatic bile flow. When NT antagonist SR48692 was given, bile flow rate decreased to the basal level. These results suggest that intestinal NT, released by lipid, may participate in the regulation of hepatic bile acid output by a mechanism requiring an intact enterohepatic circulation.     

Time course of insulin-receptor binding and insulin-induced lipogenesis in isolated rat fat cells.

1. Isolated rat fat cells were incubated at 37 degrees with [U-14C]-glucose 0.55 mM and 125I-labeled insulin. The amount of receptor-bound 125I-labeled insulin and the rate of insulin-induced 14C-lipid synthesis were assessed during association and dissociation of 125I-labeled insulin. 2. The rate of 14C-lipid synthesis was constant from zero time in the absence of insulin and in the presence of insulin in a high concentration (0.7 muM). With insulin in a low concentration (56 pM) the insulin-induced rate of 14C-lipid synthesis was proportional to the receptor occupancy; the receptor binding reached equilibrium and the rate of 14C-lipid synthesis reached a constant value after 30 to 45 min. With insulin in a concentration of 0.7 nM the rate of 14C-lipid synthesis reached a steady state before equilibrium of the receptor binding was obtained. 3. Ater preincubation with 56 pM 125I-labeled insulin followed by removal of extracellular insulin the decrease in the rate of insulin induced 14C-lipid synthesis followed the decrease in receptor occupancy with a half-time of about 10 min. After preincubation with insulin in concentrations of 0.28, 0.56, and 1.4 nM a maximum rate of 14C-lipid synthesis was maintained for about 8, 15, and 30 min, respectively. 4. The following model is suggested. Binding of insulin to the previously described receptors with a dissociation constant of about 3 nM (Gammeltoft, S., and Gliemann, J. (1973) Biochim. Biophys Acta 320, 16-32) represents the first step in the action of insulin on lipid synthesis from glucose. The receptor occupancy is rate-determining at low concentrations of insulin, i.e. when the occupancy is small (about 2 percent or less). At higher insulin concentrations some other step becomes rate-determining and the higher occupancy at equilibrium therefore causes no further increase in the steady state lipogenesis. However, a high receptor occupancy causes a prolonged maintenance of a maximal (or near-maximal) effect after removal of insulin from the medium.     

Relationship of actin polymerization and depolymerization to light scattering in human neutrophils: dependence on receptor occupancy and intracellular Ca++

When exposed to the N-formylated chemoattractant peptides, neutrophils undergo a transient ruffling followed by a polarization that involves a redistribution of F-actin (Fechheimer, M., and S. H. Zigmond, 1983, Cell Motil., 3:349-361). The cells also undergo a biphasic right angle light scatter response whose first phase is maximal 10-15 s after exposure to the stimulus, and whose second phase is longer in duration and maximal only after 1 min or more (Yuli, I., and R. Snyderman, 1984, J. Clin. Invest. 73:1408-1417). We now report that the first phase is accompanied by a transient polymerization of actin (monitored by cytometric analysis of phallacidin staining according to the method of Howard, T. H., and W. H. Meyer, 1984, J. Cell Biol., 98:1265-1271) and the second phase is accompanied by a more sustained polymerization of actin. Based on correlated measurements of ligand binding (Sklar, L. A., D. A. Finney, Z. G. Oades, A. J. Jesaitis, R. G. Painter, and C. G. Cochrane, 1984, J. Biol. Chem., 259:5661-5669) and intracellular Ca++ elevation (under conditions where we use the fluorescent Ca++ chelator Quin 2 to modulate intracellular Ca++ levels), we conclude that this first phase requires less than 100 receptors/cell (out of 50,000) and does not require the release of intracellular stores of Ca++. In contrast, the sustained polymerization requires both the occupancy of thousands of receptors (an estimated 10% of the receptors per minute) and may be somewhat sensitive to the availability of intracellular Ca++. When ligand binding is interrupted, F-actin rapidly depolymerizes with a half-time of no greater than approximately 15 s, and the transient light scatter response decays toward its initial value in parallel. Partial disaggregation of the cells follows the recovery of these responses. Based on these observations, we suggest that transient actin polymerization and transient cell ruffling give rise to transient aggregation as long as degranulation is limited.     

Ligand/receptor internalization: a spectroscopic analysis and a comparison of ligand binding, cellular response, and internalization by human neutrophils

We have compared the kinetics of the responses of neutrophils to the kinetics of ligand-receptor interaction and internalization, using as a model ligand the fluoresceinated hexapeptide N-CHO-Nle-Leu-Phe-Nle-Tyr-Lys-Fluorescein (Nle, norleucine). Cellular responses, ie, membrane depolarization, enzyme (elastase) secretion, and superoxide anion (O-2) generation, are all initiated within 10 sec of the exposure of cells to stimulus. In the cases of membrane depolarization and secretion (in cytochalasin B-treated cells), full responses are elicited by binding which occurs within 15 sec of peptide addition. Ligand binding and internalization have been analyzed over the same time frame with new spectroscopic techniques. The association of ligand and receptor is monitored using an antibody to fluorescein. The antibody to fluorescein specifically quenches the ligand which is in solution, but receptor-bound ligand is inaccessible to the antibody. The internalization of the receptor-bound ligand is monitored by the accessibility of the fluoresceinated peptide to quenching by an external pH change (7.4 leads to 4.0). Ligand which is either outside or on the cell surface is instantaneously quenched while intracellular peptide (or intracellular fluorescein derived from fluorescein diacetate) is only slowly quenched. No internalization is observed until 1 min after binding begins and internalization proceeds at a rate of up to 5,000 receptors/min/cell following a near optimal stimulatory ligand concentration (approximately 1 nM) while the occupied receptors are being cleared from the surface. A comparison of the kinetics of internalization and the cellular responses suggests that internalization of the ligand is too slow to be involved in the triggering of the cellular responses.     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

Occurrence of VIP and peptide HM in human pancreas and their influence on pancreatic endocrine secretion in man

By immunohistochemistry it was found that VIP- and peptide HI/peptide HM (PHI/PHM)-like immunoreactivity occurred in autonomic neurons in the human pancreas. Antisera against both VIP and PHI/PHM reacted with neuronal cells in local ganglia and these ganglia also contained PHI/PHM- and VIP-immunoreactive fibre plexuses. VIP- and PHI/PHM-positive fibres were also seen close to the Langerhans' islets. In addition, PHI/PHM- but not VIP-like immunoreactivity was observed in the endocrine cells often located in the periphery of the islets. The nature of these PHI/PHM-positive cells remains to be established. I.v. infusion of VIP at constant rates of 300 and 900 pmol/kg X h for 30 min in 6 healthy volunteers resulted in plateau values of 102 +/- 26 and 291 +/- 25 pM, respectively. These levels of VIP which are above those found in the circulation under physiological conditions stimulated secretion of insulin, C-peptide and pancreatic glucagon dose-dependently. On the contrary prolonged (60 min) infusion of PHM in doses resulting in plasma levels up to 1340 +/- 405 pM had no effect on pancreatic hormone secretion. These findings suggest that VIP is a likely neurotransmitter in the control of endocrine pancreatic secretion while PHM has a less prominent role, if any.     

Oscillating actin polymerization/depolymerization responses in human polymorphonuclear leukocytes

Leukotriene B4 and platelet-activating factor induced a rapidly oscillating actin polymerization/depolymerization response in human polymorphonuclear leukocytes. N-Formylpeptides were deficient in the ability to induce these oscillations. Flow cytometric analysis of filamentous actin verified that all cells were synchronously responding in this cyclic manner. The hypothesis was tested that these oscillations were analogous to chemical oscillations, i.e. oscillations of intermediate species in chemical systems that are far from equilibrium (Epstein, I. R., Kustin, K., DeKepper, P., and Orban, M. (1983) Sci. Am. 248, 112). Actin polymerization/depolymerization cycles were terminated by adding receptor antagonist a few seconds after initiation of the response by agonists. Thus the oscillations did not represent chemical oscillations that hypothetically could result from a rapid jump of the intracellular milieu to a state far from equilibrium. Rather, continued occupancy of receptors and/or occupancy of new receptors was required to sustain the oscillations. This suggested that the oscillations resulted from regulated polymerization and depolymerization pathways. In simultaneous measurements of actin-associated right angle light scatter and intracellular calcium, no calcium oscillations were detected. Thus, cycles of actin polymerization/depolymerization were not regulated by calcium oscillations.     

Neutrophil degranulation detected by right angle light scattering: spectroscopic methods suitable for simultaneous analyses of degranulation or shape change, elastase release, and cell aggregation

We have used simultaneous spectrometric analysis of right angle scattering and elastase release from human neutrophils to demonstrate the similarity of these two measures of degranulation. Both responses depend on the presence of cytochalasin B, and are similar in kinetics, dose-response, and dependence on receptor occupancy at the formyl peptide receptor. This scattering response is shown to be largely independent of cell aggregation. In the absence of cytochalasin B, a rapid and transient right angle scatter response of a different character, probably associated with cell ruffling, is detected. Either right angle response can be detected by flow cytometry.     

Helix A stabilization precedes amino-terminal lobe activation upon calcium binding to calmodulin

The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)resorufin (ReAsH), which upon binding to an engineered tetracysteine motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the (1)H- (15)N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe ( K d = 0.36 +/- 0.04 microM), which results in a reduction in the rate of ReAsH binding from 4900 M (-1) s (-1) to 370 M (-1) s (-1). In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe require calcium occupancy of amino-terminal sites (K d = 18 +/- 3 microM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.     

Cardiovascular and renal effects of calcitonin gene-related peptide in hypertensive dogs

The systemic cardiovascular and renal effects of synthetic beta-human calcitonin gene-related peptide (beta-hCGRP) were examined in conscious normotensive and one-kidney one-clip (1K-1C) hypertensive dogs. beta-hCGRP was infused intravenously at 10 and 50 ng/kg/min for 75-min periods each. Mean arterial pressure did not change significantly (p greater than 0.05) in either group during low dose infusion of beta-hCGRP, but infusion of beta-hCGRP at 50 ng/kg/min produced a fall in mean arterial pressure from 140 +/- 4 to 116 +/- 6 mmHg (p less than 0.05) in the hypertensive dogs (n = 4) and from 100 +/- 4 to 78 +/- 3 mmHg (p less than 0.05) in the normotensive dogs (n = 4). Heart rates increased significantly during infusion of beta-hCGRP in both groups. Also, renal sodium and potassium excretion decreased (p less than 0.05) in the two groups at both the low and high doses of beta-hCGRP. Creatinine clearance was unchanged in normal dogs and decreased (p less than 0.05) in 1K-1C hypertensive dogs at the high rate of beta-hCGRP infusion. The clearance of p-aminohippurate increased approximately 20% (p less than 0.05) in both groups with the low dose infusion of beta-hCGRP but further increases were elicited only in the normotensive dogs in response to the elevation in the beta-hCGRP infusion rate. Plasma renin and aldosterone levels increased (p less than 0.05) above control levels during the maximum hypotensive response to beta-hCGRP infusion in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of exercise hemoconcentration and hyperosmolality on exercise responses

We investigated the effects of a decrease in plasma volume (PV) and an increase in plasma osmolality during exercise on circulatory and thermoregulatory responses. Six subjects cycled at approximately 65% of their maximum O2 uptake in a warm environment (30 degrees C, 40% relative humidity). After 30 min of control (C) exercise (no infusion), PV decreased 13.0%, or 419 +/- 106 (SD) ml, heart rate (HR) increased to 167 +/- 3 beats/min, and esophageal temperature (Tes) rose to 38.19 +/- 0.09 degrees C (SE). During infusion studies (INF), infusates were started after 10 min of exercise. The infusates contained 5% albumin suspended in 0.45, 0.9, or 3.0% saline. The volume of each infusate was adjusted so that during the last 10 min of exercise PV was maintained at the preexercise level and osmolality was allowed to differ. HR was significantly lower (10-16 beats/min) during INF than during C. Tes was reduced significantly during INF, with trends for increased skin blood flow and decreased sweating rates. No significant differences in HR, Tes, or sweating rate occurred between the three infusion conditions. We conclude that the decrease in PV, which normally accompanies moderate cycle exercise, compromises circulatory and thermal regulations. Increases in osmolality appear to have small if any effects during such short-term exercise.     

Cation-dependent stability of subtilisin

Subtilisin BPN' contains two cation binding sites. One specifically binds calcium (site A), and the other can bind both divalent and monovalvent metals (site B). By binding at specific sites in the tertiary structure of subtilisin, cations contribute their binding energy to the stability of the native state and increase the activation energy of unfolding. Deconvoluting the influence of binding sites A and B on the inactivation rate of subtilisin is complicated, however. This paper examines the stabilizing effects of cation binding at site B by using a mutant of subtilisin BPN' which lacks calcium site A. Using this mutant, we show that calcium binding at site B has relatively little effect on stability in the presence of moderate concentrations of monovalent cations. At [NaCl] =100 mM, site B is >or=98% occupied with sodium, and therefore its net occupancy with a cation varies little as subtilisin is titrated with calcium. Exchanging sodium for calcium results in a 5-fold decrease in the rate of inactivation. In contrast, because of the high selectivity of site A for calcium, its occupancy changes dramatically as calcium concentration is varied, and consequently the inactivation rate of subtilisin decreases approximately 200-fold as site A becomes saturated with calcium, irrespective of the concentration of monovalent cations.     

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.     

Two-phase effect of endothelin-1 on resistance of coronary vessels in anesthesized rats with intact chest]

Effects of intracoronary infused (2 pM/kg/min for 5 min) endothelin-I on coronary blood flow was studied using modification of the method of Vetterlein and Schmidt. Blood flow in extracorporeal circuit was measured by 20 MHz pulsed Doppler flowmeter. One end of the circuit was connected to the left common carotid artery and the other was connected to the especially curved glass cannule which was placed to the origin of the coronary artery and through the right common carotid artery. Five-minute infusion of endothelin was followed by transitory dilatation and then by constriction of coronary vessels. Blockade of dihydropyridine-sensitive Ca-channels potentiated endothelin-induced vasodilation and decreased the constrictor response three-fold.     

Hormone binding and coupled response relationships in systems dependent on the generation of secondary mediators

The relationship between hormone binding and biological response curves is discussed for instances in which the response is dependent upon the generation of a secondary mediator of hormone action. In the simplest model, where the rate of mediator generation is directly proportional to the level of hormone receptor occupancy, and degradation of the secondary mediator follows first-order kinetics, the form of the biological response curve is identical to that for hormone receptor occupancy, but the curve is displaced to the left such that a half-maximal biologic response occurs at less than 50% hormone receptor occupancy (spare-receptor phenomenon). The same is shown to be the case when the model is iterated to include a sequence of coupled intermediate binding reactions intervening between initial hormone binding and the final biological response. When, in contrast, the degradation of one or more of these coupled intermediates is not strictly first order, but instead shows standard Michaelis-Menten kinetics, the response curve, while again remaining parallel to the curve for hormone binding, can now move to the right of the binding curve such that a high threshold is observed for the biological effect, and a half-maximal response may not occur until the level of hormone receptor occupancy is well over 50%. Some consequences of this model are discussed with special reference to the sensitivity and speed of reversibility of the biological response, implications for responses at pharmacological as opposed to physiological concentrations of hormone, and parallels which can be extended to coupled enzymatic reactions of the Michaelis-Menten type.     

GrpE accelerates peptide binding and release from the high affinity state of DnaK

The Escherichia coli nucleotide exchange factor GrpE accelerates the rate of ADP dissociation from high affinity ADP-DnaK, thus enabling ATP binding and transition to the low affinity state. We show here that GrpE, in the absence of ATP, accelerates the rates of the forward and reverse reaction ADP-DnaK-P right harpoon over left harpoon ADP-DnaK + P, where P denotes peptide substrate. Specifically, the binding of GrpE to an ADP-DnaK-P (or DnaK-P) complex increases koff and kon by approximately 200-fold and approximately 60-fold, respectively. The results are consistent with a GrpE- induced conformational change in the C-terminal polypeptide binding domain of an ADP-DnaK molecule, which results in a unique low affinity intermediate from which peptide can dissociate. A simulation of peptide dissociation from DnaK as a function of the [ATP] / [ADP] ratio shows that GrpE induced peptide dissociation from ADP-DnaK is important at elevated cellular concentrations of ADP, which typically occur upon stress.