Warwick A. Arden

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.

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 Ca2+ channel activity and subsequent intracellular Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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. J. Neurosci. Res. 57:643–650, 1999.

Nω-nitro-L-arginine blocks the second phase but not the first phase of the endothelium-dependent relaxations induced by substance P in isolated rings of pig carotid artery

Summary: Endothelium‐dependent relaxations can be evoked by a variety of stimuli, among them substance P (SP), which is found in sensory nerve fibers supplying the adventitia‐media junction of most muscular arteries. This study determined the role of endothelium‐derived nitric oxide as a mediator of endothelium‐dependent relaxations to SP in isolated rings of the pig carotid artery suspended in organ chambers for isometric tension recording. SP (10−12‐10−7 M) caused concentration‐dependent relaxations of arteries precontracted with norepinephrine (10−7 M). The relaxations were characterized by a partially transient relaxation (phase 1) and a sustained relaxation of the artery (phase 2). The inhibitor of nitric oxide formation, N&ohgr;‐nitro‐L‐arginine (l‐NNA) methyl ester caused a gradual increase in tension, the phase I response at 3 × 10−10 to 3 × 10−7 M SP was shifted to the right, but the maximal relaxation was comparable in the presence of l‐NNA. However, the sustained relaxation after addition of substance P (phase II) was lost and tension in the presence of l‐NNA returned to a level above that induced by l‐NNA and norepinephrine (10−9 M). These results suggest that the endothelium‐dependent relaxations to SP, particularly the prolonged relaxation (phase II), are due to de novo synthesis of nitric oxide and hence fully abolished by a specific inhibitor.

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.

Comparison of capsaicin-evoked calcium transients between rat nodose and jugular ganglion neurons

The objectives of this study were to describe the size distribution of capsaicin-sensitive neurons in nodose and jugular ganglia and to determine whether there is a difference in capsaicin sensitivity between these two types of ganglia. Functional identification was made by measurement of the capsaicin-evoked calcium (Ca2+) transients in cultured vagal sensory neurons of young adult Sprague-Dawley rats using the Fura-2-based ratiometric imaging technique. In the first study series, cells on the second day of culture were perfused with capsaicin solution (10(-7) M) for 15 s, and the Ca2+ transients were continuously recorded before, during, and after the capsaicin challenge. Out of 603 viable neurons, 57.5% were capsaicin-sensitive; the percentages of capsaicin-sensitive cells in the nodose and jugular ganglia were 59.8% and 55.4%, respectively. Capsaicin sensitivity predominated in the small- and medium-sized neurons; the capsaicin-sensitive cells generally had a diameter less than 35 microm in both types of ganglia. Although the results did not indicate any differences in the size distribution of capsaicin-sensitive neurons between the two ganglia, results of our second study series showed that a near-maximal concentration of capsaicin (3 x 10(-6) M) evoked a significantly greater peak Ca2+ transient in jugular neurons (382.5 +/- 85.5 nM) than in nodose neurons (134.3 +/- 17.5 nM). In summary, our results showed that an increase in cell diameter was accompanied by a decreasing trend in percentage of capsaicin-sensitive neurons in both vagal ganglia. Capsaicin at high concentration evoked a greater peak Ca2+ transient in jugular ganglion neurons, despite no difference in the responses to KCl between these two types of ganglion neurons.

Involvement of tachykinins in endotoxin-induced airway hyperresponsiveness.

Abstract. 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 μl of saline vehicle, and those in Groups II and III with 1 mg of LPS (Escherichia coli, 0111:B4) in 100 μl 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 μg/kg, intravenously) and capsaicin (0–1.6 μg/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, ΔRL caused by histamine (8 μg/kg) was significantly higher in Group II (1.145%) than that in Group I (280%; p < 0.05); similarly, ΔRL caused by capsaicin (1.6 μg/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 μg/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.

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.

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 Ca2+. 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 Ca2+ 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 Ca2+ transients. At 100 nM PMA, peak Ca2+ 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 Ca2+ transients were also reduced by 20 min PMA treatment (Δ −27% ± 4%; P < 0.05), suggesting that PKC affects voltage‐gated Ca2+ channel activity. Pretreatment with PACAP also resulted in both time‐ and concentration‐dependent suppression of Ca2+ transients. After 20 min exposure to 1 μM 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 (Δ −56% ± 2%; P < 0.05) and PACAP (Δ−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 Ca2+ signaling and secretion in PAMC cells. J. Neurosci. Res. 59:760–766, 2000

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.

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.