Differential contractile responses of mesenteric and pulmonary artery segments to norepinephrine and phorbol ester in the septic pig.

The contractile response of isolated vascular segments was studied in Yucatan miniature swine approximately 48 hr after induction of sepsis by intraperitoneal injection of live Escherichia coli. Compared to non-septic controls, segments of the cranial mesenteric artery from septic animals showed a significantly attenuated contractile response to the adrenergic receptor agonist norepinephrine (NE). The EC50 for NE increased from 1.1 +/- 0.3 to 6.3 +/- 2.0 microM and the Emax decreased from 1,010 +/- 179 to 387 +/- 75 mg tension/mg tissue. In contrast, segments of the pulmonary artery showed no significant difference in contractility to NE between sham-operated and septic animals. Mesenteric and pulmonary artery segments from both septic and control animals exhibited similar contraction to the protein kinase C activator phorbol-12, 13-dibutyrate. This suggests that the observed hyporeactivity to NE in porcine mesenteric artery segments is not simply due to cellular damage by toxins associated with the septic state. The results also indicate that the impact of gram-negative sepsis on vascular contractile function varies between tissue from the systemic and pulmonary circulation in pigs.

In vivo and in vitro effects of endotoxin on vascular responsiveness to norepinephrine and signal transduction in the rat.

We investigated, after in vitro and in vivo exposure to gram-negative endotoxin, the altered responsiveness of rat aortic smooth muscle to catecholamines. Two hour exposure of aortic rings from normal rats to 100 ng/ml of Escherichia coli 0111:B4 endotoxin in vitro in an artificial medium supplemented with 5% fetal calf serum at 37 degrees C did not effect the basal and norepinephrine (NE)-stimulated (10 microM, 1 hr, 37 degrees C) phosphoinositide (PI) hydrolysis and isometric contractions induced by graded doses (1 nM to 10.0 microM) of NE. Increasing the incubation time with endotoxin to 18 hr did not alter the basal PI hydrolysis but significantly (P less than 0.05) decreased the NE-induced PI hydrolysis (30% inhibition) and contractile sensitivity to NE (increase of EC50 from 20.0 +/- 3.8 to 156.4 +/- 46.7 nM). Qualitatively similar results were obtained in experiments where rats were injected intravenously with buffer or an LD50 dose (10 mg/kg) of endotoxin. In these ex vivo measurements, only an 18 hr exposure to endotoxin caused significant (P less than 0.001) decreases in basal (58% inhibition) and NE-stimulated (75% inhibition) PI hydrolysis and in NE-induced isometric contractions (increase of EC50 from 11.0 +/- 3.3 to 664.1 +/- 280.0 nM). The results show that the endotoxin-induced hyporeactivity to alpha 1-adrenergic receptor stimulation 1) is markedly dependent on the length of endotoxin exposure, 2) does not require (although may be enhanced by) contact with blood cells and plasma, and 3) is paralleled by a decrease in both basal and NE-stimulated PI hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

A structure-affinity study of the binding of 4-substituted analogues of 1-(2,5-dimethoxyphenyl)-2-aminopropane at 5-HT2 serotonin receptors.

With [3H]ketanserin as the radioligand, structure-affinity relationships (SAFIRs) for binding at central 5-HT2 serotonin receptors (rat frontal cortex) were examined for a series of 27 4-substituted 1-(2,5-dimethoxyphenyl)-2-aminopropane derivatives (2,5-DMAs). The affinity (Ki values) ranged over a span of several orders of magnitude. It appears that the lipophilic character of the 4-position substituent plays a major role in determining the affinity of these agents for 5-HT2 receptors, 2,5-DMAs with polar 4-substituents (e.g. OH, NH2, COOH) display a very low affinity (Ki greater than 25,000 nM) for these receptors, whereas those with lipophilic functions display a significantly higher affinity. The results of these studies prompted us to synthesize and evaluate examples of newer lipophilic derivatives and several of these (e.g. n-hexyl, n-octyl) bind with very high (Ki values = 2.5 and 3 nM, respectively) affinities at central 5-HT2 sites. Although, 2,5-DMAs are generally considered to be 5-HT2 agonists, preliminary studies with isolated rat thoracic aorta suggest that some of the more lipophilic derivatives (e.g. the n-hexyl and n-octyl derivatives) are 5-HT2 antagonists.

Alterations in rat aortic alpha 1-adrenoceptors and alpha 1-adrenergic stimulated phosphoinositide hydrolysis in intraperitoneal sepsis.

We investigated the alterations of rat aortic alpha 1-adrenoceptors and alpha 1-adrenergic stimulated phosphoinositide (PI) metabolism in intraperitoneal sepsis. An analysis of [125I]-hydroxyethylaminotetralone (HEAT) binding to alpha 1-adrenoceptors on rat aortic membranes revealed decreased numbers of receptors without changes in affinity. The maximum number of binding sites decreased from 349 +/- 35 fmol/mg to 146 +/- 16 fmol/mg (P less than 0.05 vs. control). PI metabolism was similarly attenuated in aortae from septic rats. The norepinephrine-stimulated hydrolysis of [32P]-phosphatidylinositol-4,5-bisphosphate was significantly decreased in aortae from septic rats as was the alpha 1-adrenoceptor stimulated accumulation of [3H]-inositol monophosphate. Finally, the basal labeling of [32P]-phosphatidylinositol-4,5-bisphosphate but not of [32P]-phosphatidylinositol or [32P]-phosphatidic acid was significantly diminished. These results imply that signal transduction induced by alpha 1-adrenoceptor agonists in rat aorta is significantly altered in intraperitoneal sepsis. These findings may help define the mechanisms of depressed aortic contractility in models of sepsis and endotoxic shock.

Endotoxin-induced platelet activation in human whole blood in vitro.

The effect of purified bacterial endotoxin was studied on human platelets in vitro. In adding up to 1 microgram/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma. On the other hand; endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size. Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP. The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation. Thus, the activation of human platelets by "solubilized" endotoxin in plasma requires the presence of other blood cells. We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.

Effects of a phorbol ester on rat aortic contraction and calcium influx in the presence and absence of BAY k 8644.

Phorbol esters like phorbol-12,13-dibutyrate (PDB) exert potent contractile effects on rat aorta. In the present study, we determined whether phorbol esters alter contraction by augmenting calcium influx through calcium channels. Isometric tensions and calcium influx (using 45Ca) were measured in rat aortic rings exposed to PDB in the presence and absence of BAY k 8644, a calcium channel agonist. We found that PDB alone caused significant increases in isometric tension. The addition of 100 nM BAY k 8644 enhanced the PDB-induced rat aortic contraction which was inhibited as well as reversed by the calcium channel antagonist, nitrendipine. In the presence of PDB alone, calcium influx was significantly increased only after an extended time (30 min) of isometric contraction. In the presence of BAY k 8644, however, PDB caused a significant increase in calcium influx during shorter periods (10 min) of isometric contraction. We also found that the biologically inactive phorbol, 4-alpha-phorbol, did not potentiate the effects of BAY k 8644 on either contraction or calcium influx. These results indicate that PDB may induce rat aortic contraction via activation of protein kinase C which in the presence of a calcium channel agonist can potentiate the mobilization of calcium through nitrendipine-sensitive calcium channels.

Dose-dependent changes in the antigenicity of bacterial endotoxin exposed to ionizing radiation.

The antigenic properties of the highly purified US reference standard endotoxin (RSE) exposed to varying doses of ionizing radiation were studied with double immunodiffusion, immunoelectrophoresis and immunoblotting. Rabbit RSE antisera identified 2 distinct major antigenic components for untreated RSE: one related to the O-polysaccharide side chain ("O-antigenic specificity"), the other to the R-core. Based on a serologic cross-reactivity of R-core of RSE (Escherichia coli 0113) with the R-core of the lipopolysaccharide from E. coli 0111, the core type of E. coli 0113 was identified as coli R3. Increasing exposure of RSE to ionizing radiation progressively destroyed all antigenic reactivities: at lower doses of radiation the rate of elimination differed for the 2 antigen classes. The O-polysaccharide was more sensitive to gamma-radiation than the R-core and the O-antigenicity was lost before that of the R-core. Endotoxin molecules containing incomplete R-core (radiation-induced or mutant) did not react with the RSE antiserum.

Free platelet count and size distribution during C1q inhibition of collagen-induced platelet aggregation.

Electronic free platelet counting was more sensitive than turbidimetry to detect collagen-induced platelet activation in human platelet-rich plasma. Purified human C1q exhibited a greater inhibitory effect on collagen-induced platelet aggregation in turbidimetry than free platelet counting. Because the change from small to large platelet aggregates is responsible for the continuing increase in light transmission, C1q was likely more capable of blocking the formation of large platelet aggregates than the formation of small aggregates from single platelets. The rate of change by collagen in light transmission and free platelet count was reduced in the presence of C1q but the timing of the peak response remained the same. Electronic platelet sizing revealed that the volume of single platelets transiently increased during the turbidimetric "lag phase". The mean, mode and median volume of the remaining free platelets then decreased, suggesting a selective loss of large, functionally more active platelets and/or platelet degranulation. C1q had no effect on the volume increment during the "lag phase", but reduced the subsequent fall in the volume of free platelets.

Prostaglandins activate phosphoinositide metabolism in rat aorta.

The ability of the five prostaglandins to activate phosphoinositide (PI) metabolism and to induce contraction was studied in rat aorta. All prostaglandins (PG) tested (B2, D2, E2, F1 alpha and F2 alpha) stimulated PI hydrolysis in the range of 0.01-1,000 microM (EC50 s from 0.9 to 47 microM) and elicited contractions in the range of 0.1-100 microM (EC50 s from 0.3 to 22.1 microM). The rank order potency was PGD2 greater than F2 alpha greater than F1 alpha greater than E2 greater than B2 for PI hydrolysis and PGB2 greater than F2 alpha greater than D2 greater than E2 greater than F1 alpha for contraction. The activation of PI hydrolysis by the various prostaglandins was greater than or equal to the effect of 5-hydroxytryptamine, norepinephrine, angiotensin II and [Arg8]vasopressin. The PI hydrolytic activity of PGD2, E2 and F2 alpha correlated with their ability to induce contraction. The results with PGB2 and F1 alpha showed, however, that activation of PI hydrolysis per se is not always a sufficient or necessary condition for rat aortic contraction.

Relation of structure to function for the U. S. reference standard endotoxin after exposure to 60Co radiation.

The structure and function of the highly purified U.S. reference standard endotoxin (RSE) were studied after exposure to ionizing radiation from a 60Co source. With increasing doses of radiation, the trilaminar ribbon-like structure of untreated endotoxin exhibited focal swelling, after which only spherical particles were seen by electron microscopy. These morphological changes were paralleled by the respective loss of O-side chain repeating units and pieces of the R-core from the lipopolysaccharide molecules, as demonstrated by electrophoresis. The biologic function of the irradiated endotoxin was assessed with a variety of tests. At higher doses of radiation, a direct relation was observed between the degradation of the molecular and supramolecular structure and the loss of biologic function. At lower doses of radiation, however, there was variability among the functional assays in their rate of change with progressive irradiation of the RSE. The results suggest that the carbohydrate moiety plays an important role both in determining the supramolecular structure and in modulating certain biologic activities of bacterial endotoxins.

Monomeric and polymeric collagen-induced platelet aggregation in citrated and heparinized platelet-rich plasma.

The physical and chemical properties of Type I bovine collagens were studied in relation to their platelet aggregating activity in citrated and heparinized human platelet-rich plasmas (PRP). Despite close similarities in physical and chemical properties, significant differences were found in platelet aggregating potency between two monomeric atelocollagens. Skin atelocollagen was a potent and corneal atelocollagen was a very weak inducer of platelet aggregation in citrated and heparinized PRP. In a polymeric form, however, corneal atelocollagen was a stronger platelet aggregating agent than monomeric skin acid-soluble or atelocollagen. Removal of the telopeptides altered some of the characteristics of the platelet aggregation induced by monomeric skin collagen. The rate and maximum extent of aggregation were the same with skin acid-soluble (intact) and atelocollagens in either type of PRP, but the lag periods and aggregation times were longer in citrated and somewhat shorter in heparinized PRP with skin atelocollagen than with acid-soluble collagens. The possible mechanisms leading to the differences observed in platelet aggregating activity of collagens in different physical states and from different tissues, and their distinct platelet aggregation patterns in differently anticoagulated PRP, are discussed.

On the reactivity of corneal collagen and subcomponent C1q of the complement system with human platelets and IgG-coated latex particles.

Collagen was isolated from bovine cornea and tested for reactivity towards platelets and IgG-coated polystyrene latex particles. The corneal collagen caused a dose- and temperature-dependent platelet aggregation in all human platelet-rich plasmas studied. As little as 0 . 31 micrograms of purified corneal collagen could trigger platelet activation. Human C1q, a subcomponent of the first complement component (C1), which shares extensive chemical-structural similarities with collagen, was able to inhibit the platelet aggregation provoked by corneal collagen. This blocking effect could be, however, overcome by increasing collagen doses. In a slide method or in aggregometry both corneal collagen and C1q agglutinated IgG-coated latex particles in a dose-dependent manner. Addition of such latex particles to platelet-rich plasmas or preincubation of the particles with collagen reduced or prevented the platelet action of collagen, suggesting that due to their reactivity with collagen immune complexes may play an inhibitory role in collagen-caused platelet aggregation. The results are discussed in relation to pathological events that lead to collagenolysis and also with respect to wound healing in the injured cornea.

Interaction of subcomponent C1q and collagen with isolated platelet membranes in platelet aggregation.

Human platelet membranes were isolated by the glycerol-lysis technique. When tested by a turbidimetric method in the presence of fibrinogen and calcium ions, the membrane vesicles exhibited patterns of reactivity to collagen. As with human platelet-rich plasmas, the aggregating effects of collagen were inhibited by preincubating the membrane suspensions with hemolytically active human C1q. Upon addition to platelet-rich plasma, the isolated membranes reduced collagen-provoked platelet aggregation. This inhibitory effect was partially suspended by prior incubation of the membranes with native C1q. The results show that the receptors for collagen and C1q are preserved during the membrane isolation procedure and support the competitive nature of inhibition by C1q of collagen-induced platelet aggregation.

Activation of human platelets by antibodies to thymocytes and beta 2-microglobulin. III. Effect of cell absorptions on the platelet aggregating and lymphocytotoxic activities of HATG and SA beta 2mG.

Horse anti-human thymocyte globulin (HATG) and sheep anti-human beta-2-microglobulin globulin (SA beta 2-mG), both known to have immunosuppressive potency, were quantitatively absorbed by a number of human cells, then, tested for platelet aggregating and lymphocytotoxic reactivities. Peripheral blood lymphocytes, lymphoblastoid T- and B-cell lines, platelets and spermatozoa could remove varying amounts of both reactivities from HATG and SA beta 2mG. Daudi cells were effective in reducing the lymphocyte and some platelet reactivity of HATG but did not affect those of SA beta 2mG. Erythrocytes had no absorption capacity for either antibody. The qualitative and quantitative differences in absorbing with various cells on the platelet aggregating and lymphotoxic activities of HATG and SA beta 2mG indicate the high specificity and direct action of the antibodies on platelets and lymphocytes.

Effect of collagen-like substances (C1q, acetylcholinesterase and elastin) on collagen-induced platelet aggregation.

The effects of collagen-like proteins on platelets were studied in human platelet-rich plasmas. The C1q subcomponent of complement (human), acetylcholinesterase (electric eel), and elastin (bovine) had no platelet aggregating activity, despite compositional homologies with collagen in terms of hydroxyproline, hydroxylysine, proline and glycine contents. Upon preincubation with platelets, acetylcholinesterase was incapable of preventing the platelet aggregation triggered by collagen, whereas elastin exerted a weak and inconsistent blocking action. In turn, native C1q strongly inhibited collagen-induced platelet aggregation and 'aggregated' C1q, obtained by ultracentrifugation of freeze-thawed monomeric C1q, had a potentiating effect. These findings confirm the highly specific character of the platelet action of C1q.

Activation of human platelets by antibodies to thymocytes and beta 2-microglobulin. II. Effect of purified beta 2m on the platelet aggregating and lymphocytotoxic activities of HATG and SA beta 2mG.

Platelet aggregating (PAA) and lymphocytotoxic activities (LTA) of antibody preparations known to have immunosuppressive properties were studied in this work. In comparison to sheep anti-human beta-2-microglobulin globulin (SA beta 2mG), horse anti-human thymocyte globulin (HATG) was about twice as potent by weight in LTA than in PAA, suggesting that a considerable portion of antibodies in HATG had been generated against lymphocyte specific antigens. Evidence is presented that not only the LTA but also the PAA is a direct effect of both HATG and SA beta 2mG on lymphocytes and platelets. The specificity of the antibodies was investigated by means of purified human urinary beta-2-microglobulin (beta 2m). Beta-2-microglobulin had no detectable inhibitory effect on the LTA and, in most cases, on the PAA of HATG. Even in a highly sensitive assay system, no more than 5 to 7% of the total PAA of HATG was inhibitable by beta 2m. In contrast, both the PAA and LTA of SA beta 2mG could be inhibited by beta 2m in a time- and dose-dependent fashion. The results prove that HATG and SA beta 2mG exert their actions through different membrane components on both platelets and lymphocytes. This is consistent with our earlier observation that the PAA of HATG and SA beta 2mG may widely dissociate among various human platelet-rich plasmas. Relatively low response to anti-beta 2m in the presence of high reactivity to ALG/ATG can also be explained by varying plasma levels of beta 2m. Finally, inhibition of the PAA of SA beta 2mG by purified beta 2m provides the first model where the platelet aggregating effect on an inducer (anti-beta 2m) can quantitatively be blocked by its specific "solubilized" receptor (beta 2m) whose structure is fully understood.

Similarities and dissimilarities between the binding ability of C1q and collagen.

Based on chemical-structural similarities between C1q and collagen, we studied their activities in reactions which are typically induced in collagen or mediated by C1q. Human C1q suppressed the collagen-induced platelet aggregation in human platelet-rich plasmas. Both human C1q and a suspension in insoluble bovine collagen inhibited in time-dependent fashion the lysis of sensitized sheep erythrocytes (EA) by guinea-pig complement. They both agglutinated sheep erythrocytes (EA and EAC4) sensitized with rabbit haemolysin, mainly in the IgM type, polystyrene latex particles complexed with heat-denatured human IgG, a combination of horse, goat and sheep globulins, or deoxyribonucleoprotein. Heating of C1q and collagen (56 degrees C, 30 min), which disrupts the collagen fold into a random coil structure, almost completely abrogated all activities of C1q and considerably reduced those of collagen, suggesting that an intact triple helix is essential for their activities. In spite of their far-ranging similarities, C1q was more potent by weight in most reactions, showed evidence of a faster rate of binding to EA, and was more sensitive to heat treatment at 56 degrees C than was collagen. on the basis of the binding activities of collagen, a model is proposed according to which platelets, sensitized erythrocytes, aggregated gammaglobulins, immune complexes and deoxyribonucleoprotein might accumulate at the site of endothelial damage where blood and its components are exposed to collagenous substances. C1q is able to inhibit all these reactions, indicating that not only is C1q collagen-like in its behaviour, but that collagen also had C1q-like properties.

A physiologic regulator of collagen-induced platelet aggregation: inhibition by Clq.

Platelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with CLq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen. Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity in collagen in vivo.

Activation of human platelets by antibodies to thymocytes and beta 2-microglobulin. I. Qualitative and quantitative aspects of the platelet aggregation induced by HATG and SA beta 2mG.

The platelet effect of antibody preparations known to have immunosuppressive action was investigated by a turbidimetric method in vitro. Both horse anti-human thymocyte globulin (HATG) and sheep anti-human beta 2-microglobulin IgG (SA beta 2mG) caused the platelets to aggregate in all human platelet-rich plasmas (PRP) tested. The aggregation was usually irreversible and characterized by a sigmoid curve. The action is specific for the antibodies in HATG and SA beta 2mG because control preparations (horse normal IgG and IgG fractions of sheep normal, anti-dog IgG and anti-human IgA sera) were ineffective; further, heating (30 min at 56 degrees C) and BaSO4 or Al(OH)3 adsorption of HATG and SA beta 2mG did not alter their aggregating capability. When HATG and SA beta 2mG were added together to PRP, they induced aggregation in a simple additive manner. High antibody doses tended to decrease the extent of aggregation. The effect of platelet count on aggregation varied with both the dose level ('low' or 'high') and type (HATG or SA beta 2mG) of the inducer antibody. Using fixed submaximal doses, four main aggregation patterns could be recognized among 60 PRP: (i) high responses to both HATG and SA beta 2mG; (ii) high to HATG, low to SA beta 2mG; (iii) low to HATG, high to SA beta 2mG; and (iv) low to both. The results provide guidelines for quantitative aggregation studies with platelet antibodies and suggest that HATG and SA beta 2mG act through distinct platelet membrane components, the receptor for the latter being the best characterized protein of the mammalian cell membrane.