Beta adrenoceptor regulation of macrophage arginase activity.

BACKGROUND: Arginase, which metabolizes L-arginine within the urea cycle, is essential for production of polyamines and affects production of nitric oxide by depletion of L-arginine, the common substrate for both arginase and nitric oxide synthase. Having shown that trauma increases splenic macrophage arginase activity, we seek to define the mechanisms for this. RAW macrophage arginase activity and expression are increased by 8-bromo-cAMP in vitro. We hypothesize that since catecholamines increase cAMP, trauma-induced splenic arginase activity may be mediated by post-injury catecholamine release. METHODS: RAW 264.7 macrophage arginase activity was measured in vitro in response to 4 catecholamines with or without propranolol or lipopolysaccharide (LPS). C57BL/6 mice underwent laparotomy as a model of moderate trauma after propranolol treatment, with and without intraperitoneal Escherichia coli LPS administration as a simulated pro-inflammatory stimulus. RESULTS: Macrophage arginase activity increased in vitro in response to catecholamines or LPS (P < .05). Propranolol pretreatment blocked macrophage arginase activity induced by epinephrine (10 mumol/L) in vitro (P < .05). Trauma or LPS alone increased splenic arginase activity in vivo (P < .05). Propranolol did not alter LPS-induced splenic arginase activity but did significantly reduce trauma-induced splenic arginase activity (P < .05). CONCLUSIONS: Catecholamines alone increase macrophage arginase activity through beta-adrenoceptor activation. Increased splenic arginase activity induced by moderate trauma is decreased by beta-adrenoceptor blockade, suggesting that trauma-induced arginase activity is partly mediated by endogenous catecholamines.

Modulation of stimulus-secretion coupling in porcine adrenal chromaffin cells by receptor-mediated increases in protein kinase C activity.

Catecholamine (CAT) secretion by adrenal chromaffin cells is primarily triggered by nicotinic receptor-dependent increases in cytosolic Ca(2+). The principal aim of the present study was to determine whether pituitary adenylate cyclase activating peptide (PACAP), which is coreleased with acetylcholine from the splanchnic nerve, can modulate nicotinic receptor-dependent Ca(2+) signaling and catecholamine secretion in porcine adrenal medullary chromaffin (PAMC) cells. Activation of protein kinase C (PKC) with phorbol myristate acetate (PMA) dose- and time-dependently inhibited nicotine (NIC)-induced Ca(2+) transients. At 100 nM PMA, peak Ca(2+) levels were reduced by 27% +/- 2% (P < 0.05) and 41% +/- 3% (P < 0. 05) after 10 and 20 min exposure, respectively. The inhibitory effects of PMA were significantly reduced by preincubation with the PKC inhibitor staurosporine. KCl-induced Ca(2+) transients were also reduced by 20 min PMA treatment (Delta -27% +/- 4%; P < 0.05), suggesting that PKC affects voltage-gated Ca(2+) channel activity. Pretreatment with PACAP also resulted in both time- and concentration-dependent suppression of Ca(2+) transients. After 20 min exposure to 1 microM PACAP, NIC- and KCl-induced transients were reduced by 36% +/- 5% (P < 0.05) and 51% +/- 6% (P < 0.05), respectively. These effects could also be prevented by staurosporine pretreatment. NIC-induced CAT secretion was significantly reduced by pretreatment with both PMA (Delta -56% +/- 2%; P < 0.05) and PACAP (Delta-53% +/- 7%; P < 0.05). This suppressive effect on secretion could be prevented by pretreatment with staurosporine. These data suggest that, in addition to having direct stimulatory effects on catecholamine synthesis and secretion, PACAP can also negatively modulate nicotinic receptor-dependent Ca(2+) signaling and secretion in PAMC cells.

Stimulus-secretion coupling in porcine adrenal chromaffin cells: acute effects of glucocorticoids.

Recent studies from this laboratory have established that long-term exposure (48 hr) to glucocorticoids can modulate voltage-gated Ca(2+) channel activity and subsequent intracellular Ca(2+) transients in porcine adrenal medullary chromaffin (PAMC) cells maintained in primary culture. Consistent with many steroid hormone-mediated responses, this chronic effect of glucocorticoids probably involves increased gene expression and protein synthesis. However, there is now considerable evidence to suggest that steroids can also elicit acute, non-genomic effects. The aim of the present study was to determine whether acute exposure to glucocorticoids also affects nicotinic receptor-dependent catecholamine (CAT) secretion and Ca(2+) signaling in PAMC cells. Acute exposure to dexamethasone (DEX) dose-dependently attenuated the degree of nicotine (NIC)-induced CAT secretion, as well as the amplitude of NIC-induced intracellular Ca(2+) transients. Significant inhibition of CAT secretion occurred immediately upon addition of DEX, reached maximal levels within 5 min of exposure to DEX, and was rapidly reversible after steroid washout. The endogenous porcine glucocorticoid cortisol elicited similar effects. In contrast, DEX had no significant effect on KCl-induced CAT secretion or intracellular Ca(2+) transients. These data demonstrate that acute exposure to glucocorticoids can modulate stimulus-secretion coupling in PAMC cells and suggest that the primary site of action is the nicotinic receptor.

Involvement of tachykinins in endotoxin-induced airway hyperresponsiveness.

Inhaled endotoxin, lipopolysaccharide (LPS), has been shown to result in bronchial hyperresponsiveness (BHR) to endogenous bronchoconstrictive mediators such as histamine. To determine the role of sensory neuropeptides released from bronchopulmonary C-fibers in LPS-induced BHR, 24 guinea pigs were allocated randomly to the following four groups. Animals in Groups I and IV were challenged with intratracheal instillation of 100 microliters of saline vehicle, and those in Groups II and III with 1 mg of LPS (Escherichia coli, 0111:B4) in 100 microliters of saline. Groups III and IV also received a high dose capsaicin (HDC) treatment to deplete tachykinins from C-fibers 1-2 weeks prior to the experiment. Animals were anesthetized and paralyzed, and total lung resistance (RL) and compliance (Cdyn) were measured continuously during the experiment. Dose responses of RL and Cdyn to histamine (0-8 micrograms/kg, intravenously) and capsaicin (0-1.6 micrograms/kg, intravenously), a specific C-fiber stimulant, were obtained prior to and at 1, 2, and 3 h following LPS/saline vehicle challenge. At 2 h after LPS, delta RL caused by histamine (8 micrograms/kg) was significantly higher in Group II (1.145%) than that in Group I (280%; p < 0.05); similarly, delta RL caused by capsaicin (1.6 micrograms/kg) was also increased after LPS (Group I, 107%; Group II, 267%; p < 0.05). Although HDC treatment completely abolished the bronchomotor response to capsaicin in both Groups III and IV, it enhanced the LPS-induced BHR to histamine (8 micrograms/kg; Group III, 1.834%; p < 0.05). In conclusion, these results suggest that the role of tachykinins in LPS-induced BHR may be dependent upon the type and the route of administration of the bronchoactive substance studied.

Aortoscopy: a guidance system for endoluminal aortic surgery.

PURPOSE: The aim of this project was to evaluate the feasibility of aortoscopy for guidance of endoluminal aortic procedures and to determine whether aortoscopy has advantages over fluoroscopy in a pig model. METHODS: To establish feasibility aortoscopic guidance was used for making endoluminal aortic measurements, cannulating small arteries for arteriograpy, and placing intraaortic stents and grafts in 11 pigs. To compare aortoscopy and fluoroscopy measurements were made and stents were placed by a surgeon using only aortoscopic guidance in 10 pigs and by an interventional radiologist using only fluoroscopic guidance in 10 pigs. Postmortem dissections were performed to determine measurement and device placement accuracy. RESULTS: In the feasibility study aortoscopic measurements differed from postmortem measurements by a mean distance (+/- SD) of 1.2 +/- 0.2 mm. Stents and grafts were placed a mean of 2.3 +/- 1.9 mm distal to the most inferior renal artery with no stent covering an orifice. All attempts at cannulating spinal arteries greater than 2 mm in diameter were successful. In the comparison of aortoscopic and fluoroscopic guidance, fluoroscopic measurements differed from postmortem measurements by 2.6 +/- 2.4 mm (p = 0.223). Stents placed with aortoscopic guidance were 1.1 +/- 1.3 mm distal to the most inferior renal artery, whereas stents placed with fluoroscopic guidance were 3.4 +/- 2.5 mm distal to the most inferior renal artery (p = 0.019). CONCLUSIONS: These results demonstrate that aortoscopy is a useful guidance system for endoluminal aortic procedures and may have advantages over fluoroscopy alone.

Preincubation of endotoxin with monoclonal anti-lipid A (E5), but not in vivo treatment, inhibits circulatory dysfunction.

Monoclonal antibodies (mAb) directed against the toxic lipid A portion of lipopolysaccharide (LPS) have been shown to bind lipid A in vitro, but clinical trials of such mAbs have yielded mixed results. In 53 rats instrumented for macrocirculatory and cremaster muscle microcirculatory measurements, we examined whether E5, a murine-derived anti-lipid A mAb, could inhibit LPS-induced circulatory dysfunction when incubated with LPS in vitro or given separately in vivo prior to LPS administration. Compared with Control rats (Group I), rats infused with 10 mg/kg Escherichia coli LPS (Group II) displayed marked decreases in arterial pressure and cardiac output and marked decreases in erythrocyte velocity in second, third, and fourth order skeletal muscle arterioles. Infusion of 2 mg/kg E5 90 min prior to LPS infusion (Group III) did not improve cardiovascular performance. In contrast, incubation of LPS with either 2 mg/kg (Group IV) or 10 mg/kg (Group V) E5 prior to infusion significantly attenuated LPS-induced changes in both macrocirculatory and microcirculatory function. Further investigation of the disparity between the in vitro and in vivo neutralizing capacity of anti-lipid A mAbs may aid interpretation of the variable clinical results achieved with these preparations.

An angioscopic method for intraluminal aortic evaluation and stent placement.

PURPOSE: The purpose of this study was to develop an angioscopic technique to visualize the endoluminal surface of the aorta and to guide vascular stent placement. METHODS: A fiberoptic angioscope, fitted with a balloon at its tip, was passed via a carotid arteriotomy into the abdominal aorta of seven anesthetized pigs. Saline solution inflation of the balloon allowed for blood displacement and clear visualization of the endoluminal anatomy. After the left renal artery orifice had been identified with angioscopy, a catheter was inserted via a left femoral sheath to cannulate the orifice under direct visualization. The position of the catheter was verified angiographically. A vascular stent was loaded onto an angioplasty balloon, inserted through a right femoral arteriotomy, positioned by use of angioscopic visualization, and deployed immediately below the left renal artery orifice. RESULTS: The aortic trifurcation and the lumbar and renal artery orifices were clearly visualized in every animal. Vascular stents were placed in seven animals within an average of 3.14 +/- 1.14 mm (mean +/- SEM, range 0 to 8 mm) below the inferior rim of the left renal artery orifice. No stents were positioned above a renal artery orifice or obstructed blood flow. CONCLUSIONS: This angioscopic technique permitted detailed evaluation of aortic endoluminal anatomy and precise implantation of vascular stents. Direct endovascular visualization may facilitate other endovascular procedures, including endovascular grafting.

Effects of internal mammary artery pedicle coronary artery bypass grafting on aortic valve leaflet positioning.

Approximately 50% of all patients who require replacement of the aortic valve (AVR) also require coronary artery bypass grafting (CABG) for concomitant coronary artery disease. Internal mammary artery (IMA) pedicle grafts are being used with increasing frequency for this purpose. Since the ostia of the IMA are considerably downstream from the sinus of Valsalva we hypothesized the CABG would change the local coronary flow dynamics and possibly alter the timing of both natural and prosthetic valve opening and closing dynamics. Both IMA'S were dissected as pedicle grafts in five pigs and the animals were put on cardiopulmonary bypass. Anastomotic sites were the proximal 1/3 of the left anterior descending and proximal 1/5 of the right coronary arteries. Aortic root, left ventricular and right ventricular pressures were measured and flowmeter transducers were placed on the aortic root, the left main coronary artery, the right coronary artery, the left IMA and the right IMA for measuring flows. Echocardiographic images of the aortic valve, in the longitudinal view, were recorded with a simultaneous ECG. Time points were defined during each cardiac cycle based upon characteristic points in the native coronary hemodynamics. These were identified at 8, 14 and 22% of the cycle (valve opening) and 38, 45 and 55% of the cycle (valve closing). Calculations were made based upon each cycle being initiated with the ECG R wave peak. Significant alterations in flow patterns were identified and quantitated between native coronary and IMA grafts. Only minor changes in valve positioning were identified. These differences in natural valve leaflet position occurred at 22% and 45% of the cycle.

Effect of lipopolysaccharide on the physical conformation of the erythrocyte cytoskeletal proteins.

Red blood cell deformability is important for effective circulation in the capillaries. It is known that red cell deformability is significantly reduced during septic shock. Surface to volume ratio, physical effects of the cytoskeletal proteins and the fluidity of lipid bilayer are some of the important intrinsic factors that regulate this mechanical function. Alterations in the physical conformation of cytoskeletal proteins in septic conditions could significantly alter their function. In this study, erythrocytes in whole blood were treated with lipopolysaccharide, the outer covering of Gram-negative bacteria released during Gram-negative sepsis. Electron paramagnetic resonance spectroscopy in conjunction with a protein-specific maleimide nitroxide spin label covalently bound to cytoskeletal proteins was used to investigate the resulting changes occurring in the physical state of cytoskeletal proteins in isolated membranes. Treatment of red blood cells with a lipopolysaccharide concentration as low as 40 micrograms/mL of blood solution for 90 minutes showed a significant decrease in the relevant EPR parameter (p < 0.01) of the spin label bound to subsequently isolated membranes, suggestive of a decreased segmental motion of the spin label and an increase in cytoskeletal protein-protein interactions. These results suggest a marked conformational alteration in the cytoskeletal proteins induced by the lipopolysaccharide and may explain, in part, the marked reduction in red blood cell deformability during septic shock. Bacterial lipopolysaccharide does not exert most of its effects on the host directly, but rather elicits the production of host factors that leads to complex septic shock. Leukocytes, endothelial tissue and many other cells release these mediators. Leukocytes are thought to be a particularly important source of such mediators, including cytokines (tumor necrosis factor, interleukins, etc.), oxygen free radicals, proteases, and hydrolyses. In order to characterize the possible mechanism by which the lipopolysaccharide acts on the physical state of the erythrocyte cytoskeleton, erythrocytes void of leukocytes and plasma were treated with lipopolysaccharide. The relevant EPR parameter showed no significant change over the control value. These results indicate that the leukocytes and their factors are responsible for the rearrangements seen in the cytoskeletal proteins of the erythrocyte membrane.

Effect of cardiogenic shock on pharmacokinetics and tissue distribution of ampicillin-sulbactam.

A reversible cardiogenic shock model in pigs investigated shock-induced changes in the pharmacokinetics and tissue distribution of ampicillin-sulbactam and the efficacy of this antibiotic regimen in eliminating enteric bacterial translocation. Sixteen pigs were randomly allocated to 3 groups: group I (shock, ampicillin-sulbactam, n = 6), group II (no shock, ampicillin-sulbactam, n = 6), and group III (shock, no ampicillin-sulbactam, n = 4). Nalidixic acid-resistant E. coli (60 x 10(6) CFU) were instilled into a jejunal loop created in each pig, and bacterial cultures were taken from thoracic duct lymph, periportal, and mesenteric lymph nodes. Ampicillin-sulbactam was administered intravenously at a standard dose of 3 g. Results showed that 1) ampicillin and sulbactam concentrations generally increase during cardiogenic shock; 2) cardiogenic shock does not increase ampicillin concentrations in jejunum and liver; 3) during resuscitation, thoracic duct lymph ampicillin concentrations decrease; and 4) during and immediately after cardiogenic shock, standard doses of ampicillin-sulbactam appear efficacious in eliminating translocated bacteria.

Coronary vascular and myocardial effects of substance P in hypercholesterolemic rabbits.

Age-matched male New Zealand white rabbits (n = 16) were allocated to two groups: group 1 (n = 8) received a standard rabbit diet; group 2 (n = 8) received a 2% cholesterol-enriched diet. After 8 weeks of prescribed diet, hearts were excised and placed on a constant perfusion pressure Langendorff-type apparatus. Coronary flow, left ventricular pressure, and isovolumic dP/dt were continuously measured. Baseline recordings were made and then a single 5 nmol bolus dose of substance P was delivered into the coronary perfusate. Mean serum cholesterol levels in group 1 were 53 +/- 17 (SEM) mg.dl-1, in group 2 1438 +/- 143 mg.dl-1. In group 1, the injection of substance P caused mean coronary flow to increase 39 +/- 6%, mean coronary vascular resistance to decrease 28 +/- 3%, and mean dP/dt to increase 11 +/- 4%. In group 2, coronary flow increased 57 +/- 13%, coronary vascular resistance decreased 33 +/- 5%, and dP/dt increased 17 +/- 4%. Within groups, values changed significantly from baseline but these changes were not significantly different between groups. The duration of coronary flow response was 113 +/- 20 s in group 1 and 63 +/- 8 s in group 2. Substance P is a potent dilator of coronary resistance vessels and has positive inotropic effects in the rabbit. High levels of cholesterol exposure do not alter the magnitude of substance P-induced vasodilation, but the duration of the response is shortened.

Skeletal muscle microcirculatory response to rat alpha-calcitonin gene-related peptide.

We used in vivo video microscopy to determine the effect of increasing doses of rat alpha-calcitonin gene-related peptide (rCGRP) on rat cremaster muscle arterioles in the presence or absence of the nitric oxide synthase inhibitor N-omega-nitro-L-arginine (L-NNA). Male Sprague-Dawley rats (118-148 g) were anaesthetized with pentobarbital, and neurovascularly intact cremaster muscles were imaged. Changes in the diameter, erythrocyte velocity and volume flow in second-(A2), third-(A3), and fourth-(A4) order arterioles were determined. To produce uniform arteriolar tone, the cremaster preparation was challenged with norepinephrine (NE: 10(-7) M). L-NNA (10(-4) M), which was shown to inhibit acetylcholine-(ACh: 10(-6) M) induced arteriolar dilations, was added to 16 of the preparations. Preparations were then challenged by adding cumulative log concentrations of rCGRP (10(-12)-10-7) M; n = 16) or an equivalent volume of vehicle (n = 19) to the bath. Following rCGRP challenge, arterioles were maximally dilated with 10(-5) M nitroprusside (NP). rCGRP caused significant dose-dependent increases in erythrocyte velocity and volume flow in A2 arterioles, and in diameter, velocity, and volume flow in A3 and A4 arterioles, by 10(-8) M, when compared with vehicle-treated controls. L-NNA had no significant effect on rCGRP-induced responses. These data indicate that rCGRP causes dose-dependent dilation of skeletal muscle resistance arterioles at a concentration similar to that observed in larger vessels. This dilation does not appear to be dependent on the vascular production of nitric oxide from L-arginine.

Early endotoxic shock results in enhanced vasodilator responses to nitroglycerin but unaltered responses to neuropeptides calcitonin gene-related peptide and substance P.

To determine the role that vasoactive neuropeptides, calcitonin gene-related peptide, and substance P play in tissue-blood flow regulation during early septic shock, we examined the responsiveness of arteries removed from pigs 3 h after administration of Escherichia coli lipopolysaccharide or saline vehicle. The carotid, cranial mesenteric, and left anterior descending coronary arteries were excised, and rings were cut from each vessel. Constrictor responses were obtained to cumulative doses of norepinephrine or potassium chloride. Rings were reconstricted and challenged with acetylcholine, substance P, calcitonin gene-related peptide, and nitroglycerin. Lipopolysaccharide significantly increased the cranial mesenteric artery's response to high concentrations of norepinephrine and the response to nitroglycerin in all vessels. This enhancement of responses to nitroglycerin suggests augmented smooth-muscle responsiveness to an exogenous source of nitric oxide, possibly associated with early depression of basal endothelial function. Depression of agonist-induced nitric oxide release may mask such enhancement with endothelial-dependent dilators and may enhance the response to adrenergic constrictors in some vascular beds.

Substance P induces biphasic endothelium-dependent relaxations in pig and rabbit carotid arteries.

Careful handling and preparation of freshly harvested vessels from 22 pigs and 12 rabbits revealed a two-phase vasorelaxation response to cumulative doses of substance P (SP). A rapid, transient relaxation was observed during the cumulative dose-response and a new plateau of equilibrium was seen following an increase in developed force after the last dose of SP. The phase 2 response is also produced by submaximal doses of SP and is not altered by pretreatment of the rings with Indomethacin. Acetylcholine (ACh) caused an endothelium-dependent relaxation but without evidence of a phase 2 plateau. N omega-Nitro-L-Arginine (L-NNA) and N omega-Nitro-L-Arginine Methylester (L-NAME) pretreatment resulted in a shift to the right in the phase 1 response to SP and a complete blockade of phase 2. Methylene blue caused nearly complete block of both phases. Nitroglycerin caused a dose-dependent and prolonged vasorelaxation with no phase 2.

Small intestinal microcirculatory effects of octreotide.

Somatostatin and its analogue, octreotide acetate, are thought to decrease mesenteric blood flow; however, it is unknown whether the decrease occurs at the central, regional, or microvascular level. We hypothesized that the circulatory effects of octreotide are regulated at the microvascular level. Changes in superior mesentery artery (SMA) flow in response to octreotide were measured with a perivascular ultrasonic flow probe. In separate experiments, the jejunal microcirculatory effects of octreotide were studied using in vivo videomicroscopy. After accrual of baseline hemodynamic and microcirculatory data, animals were randomized to control or treatment (10 micrograms/kg octreotide) iv groups. Measurements were made every 15 min during the infusion and for 90 min after the completion of the infusion. Results are expressed as means +/- SEM. Intravenous infusion of octreotide caused no significant change in arterial pressure, cardiac index, or systemic vascular resistance index in either group in either set of experiments. A statistically significant decrease in heart rate (9%) occurred in the control group of animals undergoing SMA flow measurement. SMA flow did not change significantly with infusion of octreotide. In contrast, jejunal first-order arteriole flow increased to 117.9 +/- 9.7% of baseline (P < 0.05) in the absence of significant changes in microvessel diameters. This was due to an increase in centerline red cell velocity (116 +/- 5% of baseline, P < 0.05). We conclude that octreotide increases jejunal first order arteriole flow by mechanisms that are regulated at the microcirculatory level.

Elevations in circulating calcitonin gene-related peptide correlate with hemodynamic deterioration during endotoxic shock in pigs.

Calcitonin gene-related peptide (CGRP) is a potent vasodilatory neuropeptide, which may play a role in vascular dysfunction during septic shock. Sixteen pigs (25-50 kg) were anesthetized with ketamine and isoflurane in O2, and administered 100 micrograms/kg Escherichia coli lipopolysaccharide i.v. (LPS; n = 8) or saline vehicle (n = 8). Pigs were instrumented for hemodynamic determinations and blood sampling for CGRP assay (pg/ml) from the portal vein (PV) and the pulmonary (PA) and carotid (CA) arteries. Blood samples were collected into EDTA and aprotinin before (baseline) and at 60, 120, and 180 min after LPS administration. LPS caused significant deterioration in indices of hemodynamic function and a significant increase in plasma CGRP concentration at all sampling sites by 120 min (P < 0.01). No significant difference between sampling sites was recorded at any time. Plasma CGRP concentrations displayed significant negative correlations with mean arterial pressure, cardiac index, and left ventricular stroke work. These data confirm our previous findings of CGRP elevations in endotoxemic rats, and indicate that 1) LPS is a potent stimulus for the systemic release of CGRP, 2) increasing plasma CGRP concentrations temporally correlates with cardiovascular deterioration during LPS shock, and 3) there is little evidence that the portal circulation is a major source of circulating CGRP levels during LPS shock. Vasoactive neuropeptides, such as CGRP, may interact with other documented mediators of vascular dysfunction in the pathogenesis of septic shock.

Nitroglycerin (exogenous nitric oxide) substitutes for endothelium-derived nitric oxide in potentiating vasorelaxations and cyclic AMP elevations induced by calcitonin gene-related peptide (CGRP) in rat aorta.

Rat calcitonin gene-related peptide (rCGRP) causes endothelium-dependent vasorelaxations via a dual signal transduction mechanism involving elevations of both cyclic AMP and cyclic GMP levels in rat aorta. These responses are all dependent on de novo synthesis of nitric oxide (NO) in endothelial cells and appear to involve a mechanistic link between cyclic GMP and cyclic AMP responses in smooth muscle cells. The present study determined whether NO from an exogenous source (i.e. added nitroglycerin) could substitute for endogenous NO in rCGRP-induced responses in endothelium-denuded aorta. Nitroglycerin (1 microM) significantly elevated cyclic GMP levels by 20-fold and 3.3-fold and cyclic AMP levels by 26% and 22% at 1 and 2 min, respectively. By itself, rCGRP (100 nM) did not significantly elevate cyclic AMP levels. In combination, however, nitroglycerin and rCGRP caused more-than-additive cyclic AMP elevations (41% above basal at 1 and 2 min). Nitroglycerin also potentiated rCGRP-induced vasorelaxations in endothelium-denuded rings, thus uncovering a direct (endothelium-independent) relaxant effect of rCGRP in rat aorta. The data indicate that exogenous NO can substitute for endogenous NO in rCGRP-induced relaxant and cyclic AMP responses in aorta. This nitroglycerin-induced potentiation of CGRP effects likely involves inhibition of cyclic-GMP-inhibited-phosphodiesterase in smooth muscle cells, thus allowing cyclic AMP to accumulate and mediate the direct vasodilator effects of rCGRP.

Effect of endotoxin on lipid order and motion in erythrocyte membranes.

Electron paramagnetic resonance employing a lipid-specific spin label has been used to investigate the molecular effects of endotoxin on the physical state of bilayer lipids in rat erythrocyte membranes. When added at a concentration as low as 40 micrograms/ml to whole blood (plasma plus leukocytes present), decreased membrane lipid motion was found in subsequently washed and spin-labeled intact erythrocytes (P < 0.02). However, if endotoxin were added to washed, plasma plus leukocyte-free intact erythrocytes, no change in the motion of the spin label was found, suggesting that plasma-soluble substances and/or leukocytes are required to produce the change in the physical state of lipids. The decreased lipid motion found in these studies is discussed with reference to the known decreased deformability of endotoxin-treated red cells and to the pathogenesis of sepsis.

Effect of anti-lipid A monoclonal antibody (E5) on microcirculatory function during lipopolysaccharide shock.

Early septic shock is characterized by fever, increased cardiac output, decreased systemic vascular resistance, and dilation of higher-order arterioles in peripheral tissues, such as skeletal muscle. We used a rat model of low-dose lipopolysaccharide (LPS) "septic" shock to investigate the potential benefit of an anti-lipid A monoclonal antibody preparation (E5) on macro- and microcirculatory function. Twenty-five male Sprague-Dawley rats were anesthetized and instrumented for measurement of arterial pressure (AP), heart rate (HR), and cardiac output (CO). The left cremaster muscle of each rat was prepared for in vivo video microscopic examination of changes in third-order arteriolar (A3) diameter and erythrocyte velocity. Rats were randomly assigned to two groups: Group I (n = 13) received E5 vehicle and 200 micrograms/kg Escherichia coli LPS; Group II (n = 12) received 2 mg/kg E5 iv prior to LPS administration. All variables were recorded at 15-min intervals for 30 min prior to and 150 min following LPS. Microcirculatory recordings were restricted to those rats where arteriolar diameters were 20-40 microns and vessels displayed obvious vasomotion (n = 7/group). Infusion of LPS caused no significant change in AP, an increase in CO by 105 min, an increase in HR by 75 min, an increase in diameter by 75 min, and a decrease in velocity by 165 min (P < 0.01). Pretreatment with E5 inhibited the A3 vasodilation but did not affect the macrocirculatory changes. These data suggest a potential therapeutic role for E5 in ameliorating LPS-induced changes in skeletal muscle microcirculation.

Scintigraphic evaluation of bacterial translocation during hemorrhagic shock.

We investigated the use of gamma scintigraphy to evaluate the temporal and spatial patterns of translocation of radiolabeled Escherichia coli from the porcine jejunum during and following hemorrhagic shock. Thirteen healthy mixed breed pigs (22-43 kg) were randomly allocated to two groups. Pigs were anesthetized with sodium pentobarital (30 mg/kg) and mechanically ventilated (100% O2). Each pig was instrumented for mean arterial pressure (MAP) and superior mesenteric artery (SMA) blood flow determination. A 25-cm loop of vascularly intact distal jejunum was isolated, and 10 mCi (10(11) cfu) of radiolabeled E. coli (99mTcO4-) was placed within the bowel segment. Consecutive 5-min scintigrams of the entire abdomen and thorax were collected for 6 hr. Pigs in the shock group (n = 7) were hemorrhaged such that MAP was maintained at 50-60 mm Hg for 5 hr. Pigs in the sham group (n = 6) were maintained without hemorrhage for 6 hr. The total radioactive counts in the translocation regions of the scintigram were plotted against time, and the slope of the regression lines was compared between groups. In the shock group, SMA blood flow decreased significantly (P < 0.05) during the hypotensive period but returned above baseline during reperfusion. The mean (+/- SD) slopes for translocation regression lines were 9.3 +/- 11.4 counts/min and 36.3 +/- 33.7 counts/min in the sham and shock groups, respectively (P < 0.05). Translocation was scintigraphically evident 50-100 min following induction of hemorrhage and did not require reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)