Optimization of the Promega PowerSeq™ Auto/Y system for efficient integration within a forensic DNA laboratory.

The application of massively parallel sequencing (MPS) is growing in the forensic DNA field, as forensic DNA laboratories are continuously seeking methods to gain information from a limited or degraded forensic sample. However, the laborious nature of current MPS methodologies required for successful library preparation and sequencing leave opportunities for improvement to make MPS a practical option for processing forensic casework. In this study, the Promega PowerSeq™ Auto/Y System Prototype, a MPS laboratory workflow that incorporates multiplex amplification, was selected for optimization with the objectives to introduce automation for relieving manual processing, and to reduce the number of steps recommended by the standard protocol. Successful changes in the optimized workflow included a switch from column-based PCR purification to automatable bead-based purification, adoption of the library preparation procedures by a liquid handling robot platform, and removal of various time-consuming quality checks. All data in this study were found to be concordant with capillary electrophoresis (CE) data and previously-generated MPS results from this workflow. Read abundance and allele balance, metrics related to sample interpretation reliability, were not significantly different when compared to samples processed with the manufacturer's protocol. All the modifications implemented resulted in increased laboratory efficiency, reduced the protocol steps associated with risk of contamination and human error events, and decreased manual processing time by approximately 12h. These findings provide forensic DNA laboratories a more streamlined option when considering implementation of a MPS workflow.

Blockade of MUC1 expression by glycerol guaiacolate inhibits proliferation of human breast cancer cells.

We sought to determine whether administration of glycerol guaiacolate at an optimal biological dose inhibits human breast cancer cell growth. Human breast cancer MCF-7 and ZR-75-1 cells were treated with glycerol guaiacolate and the therapeutic efficacy and biological activity of this drug was investigated on breast cancer cell growth. MCF-7 cells were injected into the mammary fat pad of overectamized female athymic nude mice. Ten days later, animals were treated with daily intraperitoneal injections of glycerol guaiacolate for six weeks. Tumor size and volume was monitored and immunohistochemistry analysis on MUC1, p21 and ki-67 was performed. Glycerol guaiacolate decreased breast cancer cell growth in a dose-dependent manner, decreased cell migration, and caused G1 cell cycle arrest. Our results demonstrate that glycerol guaiacolate inhibits MUC1 protein and mRNA expression levels and significantly increased p21 expression in human breast cancer cells as well as induced PARP cleavage. Similarly, glycerol guaiacolate inhibited breast tumor growth in vivo as well as enhanced p21 expression and decreased breast tumor cell proliferation (ki-67 expression). Collectively, our results demonstrate that glycerol guaiacolate decreased MUC1 expression and enhanced cell growth inhibition by inducing p21 expression in breast cancer cells. These findings suggest that glycerol guaiacolate may provide a novel and effective approach for the treatment of human breast cancer.

Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation.

BACKGROUND: Tissue factor (TF)-bearing microparticles (MP) from different origins are thought to be involved in the pathogenesis of cancer-associated thrombosis. However, the role of circulating tumor cell-derived TF is not well understood. METHODS: TF antigen and activity were measured in MP generated in vitro from human TF-expressing cancer cells by ELISA and clotting or thrombin generation assays, respectively. TF antigen and activity were also measured in vivo in cell-free plasmas from mice previously injected with in vitro-generated MP or in cell-free plasmas from nude mice bearing orthotopically injected human cancer cells. RESULTS: Tumor cell-derived MP (TMP) exhibited strong TF-dependent procoagulant activity (PCA) in vitro and in vivo. Injection of TMP into mice was associated with acute thrombocytopenia and signs of shock, which were prevented by prior heparinization. Human TF antigen and activity could be detected in mouse cell-free plasmas up to 30 min after TMP injections. Human TF was detected in the spleen of injected mice and its clearance from circulation was delayed in splenectomized mice, suggesting the involvement of the spleen in the rapid clearance of circulating MP in vivo. Detectable levels of TF-dependent PCA and thrombin-antithrombin complex were found in cell-free plasmas from mice growing pancreatic human tumors, suggesting that circulating tumor-derived TF causes coagulation activation in vivo. CONCLUSIONS: MP derived from certain cancer cells exhibit TF-dependent PCA both in vitro and in vivo. These results provide new information about the specific contribution of tumor-derived MP to the hypercoagulable state observed in cancer.

Radioiodine therapy of colon cancer following tissue-specific sodium iodide symporter gene transfer.

We investigated the feasibility of using radioiodine therapy in colon carcinoma cells (HCT 116) following tumor-specific expression of the human sodium iodide symporter (hNIS) using the carcinoembryonic antigen (CEA) promoter. HCT 116 cells were stably transfected with an expression vector, in which hNIS cDNA has been coupled to a CEA promoter fragment. This promoter is responsible for tissue-specific expression of CEA in gastrointestinal tract epithelium, and has been shown to target therapeutic genes to colorectal cancer cells. Functional NIS expression was confirmed by iodide uptake assay, Western blot analysis, immunostaining and in vitro clonogenic assay. The stably transfected HCT 116 cells concentrated (125)I about 10-fold in vitro without evidence of iodide organification. In contrast, transfection of control cancer cells without CEA expression did not result in iodide accumulation. Western blot analysis using a hNIS-specific antibody revealed a band of approximately 90 kDa. In addition, immunostaining of stably transfected HCT 116 cells revealed hNIS-specific membrane-associated immunoreactivity. In an in vitro clonogenic assay approximately 95% of stably transfected HCT 116 cells were killed by exposure to (131)I, while only about 5% of NIS-negative control cells were killed. Further, using an adenovirus carrying the NIS gene linked to the CEA promoter, high levels of tumor-specific radioiodide accumulation were induced in HCT 116 cells. In conclusion, a therapeutic effect of (131)I has been demonstrated in colon carcinoma cells following induction of tumor-specific iodide uptake activity by CEA promoter-directed NIS expression in vitro. This study demonstrates the potential of NIS as a therapeutic gene allowing radioiodine therapy of colon cancer following tumor-specific NIS gene transfer.

The sodium-iodide symporter.

The sodium-iodide symporter (NIS) is an intrinsic plasma membrane protein that mediates active transport of iodide in the thyroid gland and several other extra-thyroidal tissues. This activity has been utilized for many years for imaging the thyroid gland and for treatment of thyroid disease both benign and malignant. Cloning and characterization of NIS has more recently allowed research into its use in non-thyroidal cancers through gene transfer for both diagnosis and treatment.

Amino acid substitution at position 99 affects the rate of CRP subunit exchange.

We investigated the characteristics of CRP having amino acid substitutions at position 99. Analysis of amino acid residue proximity to cAMP in molecular dynamics (MD) simulations of the CRP:(cAMP)(2) complex [García, A. E., and Harman, J. G. (1996) Protein Sci. 5, 62-71] showed repositioning of tyrosine 99 (Y99) to interact with the equatorial exocyclic oxygen atom of cAMP. To test the role of Y99 in cAMP-mediated CRP activation, Y99 was substituted with alanine (A) or phenylalanine (F). Cells that contained the WT or mutant forms of CRP induced beta-galactosidase in the presence of cAMP. Purified WT, Y99A, and Y99F CRP showed only a 3- to 4-fold difference in cAMP affinity. There were no apparent differences between the three forms of CRP in cAMP binding cooperativity, in CRP:(cAMP)(1) complex binding to lacP DNA, in the formation of CRP:cAMP:RNAP complexes at lacP, or in CRP efficacy in mediating lacP activity in vitro. The apo-form of Y99A CRP was more sensitive to protease than the apo-form of either WT CRP or Y99F CRP. Whereas the WT or Y99F CRP:(cAMP)(1) complexes were cleaved by protease at hinge-region peptide bonds, the Y99A CRP:(cAMP)(1) complex was cleaved at peptide bonds located at the subunit interface. The rates of subunit exchange for Y99A CRP, both in the apo-form and in a 1:1 complex with cAMP, were significantly greater than that measured for WT CRP. The results of this study show that tyrosine 99 contributes significant structural stability to the CRP dimer, specifically in stabilizing subunit association.

Optimization for the blockade of epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma.

We determined the optimal administration schedule of a novel epidermal growth factor receptor (EGFR) protein tyrosine kinase inhibitor (PKI), PKI 166 (4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine), alone or in combination with gemcitabine (administered i.p.) for therapy of L3.6pl human pancreatic carcinoma growing in the pancreas of nude mice. Seven days after orthotopic implantation of L3.6pl cells, the mice received daily oral doses of PKI 166. PKI 166 therapy significantly inhibited phosphorylation of the EGFR without affecting EGFR expression. EGFR phosphorylation was restored 72 h after cessation of therapy. Seven days after orthotopic injection of L3.6pl cells, groups of mice received daily or thrice weekly oral doses of PKI 166 alone or in combination with gemcitabine. Treatment with PKI 166 (daily), PKI 166 (3 times/week), or gemcitabine alone produced a 72%, 69%, or 70% reduction in the volume of pancreatic tumors in mice, respectively. Daily oral PKI 166 or thrice weekly oral PKI 166 in combination with injected gemcitabine produced 97% and 95% decreases in volume of pancreatic cancers and significant inhibition of lymph node and liver metastasis. Daily oral PKI 166 produced a 20% decrease in body weight, whereas treatment 3 times/week did not. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased tumor cell and endothelial cell apoptosis correlated with therapeutic success. Collectively, our results demonstrate that three weekly oral administrations of an EGFR tyrosine kinase inhibitor in combination with gemcitabine are sufficient to significantly inhibit primary and metastatic human pancreatic carcinoma.

Inhibition of growth and metastasis of human pancreatic cancer growing in nude mice by PTK 787/ZK222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases.

Since vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis, we determined whether blockage of VEGF receptor signaling using a novel tyrosine kinase inhibitor (PTK 787) decreases the growth and metastasis of human pancreatic carcinoma growing orthotopically in nude mice. Human pancreatic L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice were given daily oral administrations of PTK 787 alone, twice weekly i.p. injections of gemcitabine, or combination therapy. The mice were necropsied when control mice became moribund (day 35). Therapy with PTK 787 alone, gemcitabine alone, or the combination of both agents produced respectively 60%, 70%, and 81% inhibition in the volume of pancreatic cancers. The combination therapy significantly decreased the incidence of lymph node and liver metastasis, leading to a significant increase in survival. Microvessel density (MVD) was significantly decreased in tumors treated with either PTK 787 alone or PTK 787 plus gemcitabine. MVD directly correlated with tumor cell proliferation and inversely correlated with apoptosis of tumor cells and associated endothelial cells. Collectively, our results demonstrate that blockade of VEGF-R signaling may provide an additional approach to the therapy of pancreatic cancer.

Position 127 amino acid substitutions affect the formation of CRP:cAMP:lacP complexes but not CRP:cAMP:RNA polymerase complexes at lacP.

The lacP DNA binding and activation characteristics of CRP having amino acid substitutions at position 127 were investigated. Wild-type (WT) and T127C CRP footprinted lacP DNA in the presence of DNase I in a cAMP-dependent manner. The T127G, T127I, and T127S forms of CRP failed to footprint lacP both in the absence and in the presence of cAMP. Consistent with these data, WT and T127C CRP:cAMP complexes exhibited high affinity for the lacP CRP site whereas T127G, T127I, or T127S CRP:cAMP complexes exhibited low affinity for the lacP CRP site. CRP:cAMP:RNA polymerase (RNAP) complexes formed at lacP in reactions that contained WT, T127C, T127G, T127I, or T127S CRP. These results demonstrate that allosteric changes important for cAMP-mediated CRP activation are differentially affected by amino acid substitution at position 127. Proper cAMP-mediated reorientation of the DNA binding helices required either threonine or cysteine at position 127. However, cAMP-dependent interaction of CRP with RNAP was accomplished regardless of the amino acid at position 127. RNAP:lacP complexes that supported high-level lac RNA synthesis formed rapidly in reactions that contained WT or T127C CRP whereas RNAP:lacP complexes that supported only low-level lac RNA synthesis formed at slower rates in reactions that contained T127I or T127S CRP. The T127G CRP:cAMP:RNAP:lacP complex failed to activate lacP. The results of this study lead us to conclude that threonine 127 plays an important role in transduction of the signal from the CRP cyclic nucleotide binding pocket that promotes proper orientation of the DNA binding helices and only a minor, if any, role in the functional exposure of the CRP RNAP interaction domain.

Calcium-activated potassium channel-mediated arteriolar relaxation during endotoxic shock.

The role of calcium-activated potassium (KCa) channels in the in vivo relaxation of arterioles was investigated before endotoxin shock (Pre-ENDT) and during endotoxin shock at 180 min (Post-ENDT). Diameters of 2nd and 3rd order (A2 and A3) arterioles in the left cremaster muscle of male Sprague-Dawley rats anesthetized with pentobarbital sodium were measured using videomicroscopy. Adenosine (ADO) at 534 micrograms intraarterially, topical ADO at 10(-3) M, and the endothelium-dependent agonist topical acetylcholine (ACH) at 10(-4) M significantly dilated both A2 and A3 arterioles Pre-ENDT and Post-ENDT. Topical tetraethylammonium chloride (TEA) at 1 mM blocked ADO (intraarterially and topical)-induced A2 and A3 arteriolar dilations Pre-ENDT and Post-ENDT. Arteriolar dilation to ACH was maintained Pre-ENDT, but was blocked by TEA in A2 and A3 arterioles Post-ENDT. The endothelium-independent agonist sodium nitroprusside (10(-5) M), when topically applied, caused maximal arteriolar dilation Pre-ENDT and Post-ENDT in the presence of TEA. The data show that vascular smooth muscle KCa channels are a significant factor in ADO-induced relaxation of cremaster microvessels and are not significantly affected by ENDT. The results also suggest that the mechanism for endothelium-dependent ACH vasodilation changes from a non-KCa channel-mediated mechanism Pre-ENDT to a KCa-mediated mechanism Post-ENDT.

Molecular cloning of a Schizosaccharomyces pombe cDNA encoding lanosterol synthase and investigation of conserved tryptophan residues.

A Schizosaccharomyces pombe cDNA encoding lanosterol synthase was cloned by complementing a Saccharomyces cerevisiae lanosterol synthase mutant. The predicted 83-kDa protein is 54-58% identical to other lanosterol synthases. The previously known lanosterol synthases contain 229 conserved residues, which should encompass the catalytically essential amino acids. This number is decreased dramatically by including the Sc. pombe lanosterol synthase in the analysis; 42 residues are no longer conserved and therefore are catalytically nonessential. We have begun mutagenic studies to identify catalytic residues from the remaining conserved residues. Mutant Sa. cerevisiae lanosterol synthase genes were generated in which phenylalanine was specifically substituted for conserved tryptophan residues. All of the resultant mutant enzymes retained the ability to complement the Sc. cerevisiae lanosterol synthase mutant, suggesting that these conserved tryptophan residues are not catalytically essential.

Arteriolar reactivity of endotoxin-tolerant rats after hemorrhage and reinfusion.

Adrenergic and endothelium-dependent arteriolar reactivity are greatly reduced in hemorrhagic shock. However, development of tolerance to endotoxin may prevent the decrease. The reactivity of cremaster muscle arterioles was tested in pentobarbital-anesthetized endotoxin-tolerant (ENDT-T) and nontolerant control rats. Tolerance was developed by sublethal intraperitoneal injections of Escherichia coli endotoxin for 4 days (n = 9). Controls received saline (n = 9). Mean arterial pressure (MAP), arteriolar diameter-response curves to topical norepinephrine (NE) (10-9M to 10-3M) and responses to 10-3M acetylcholine (ACh) were obtained as follows 1) at control, 2) following hemorrhage to 40 mmHg. 3) after uptake of 25% of bled volume with the remainder infused, and 4) at 240 min post-hemorrhage. The A1, A2, and A3 arterioles were constricted following hemorrhage in the ENDT-T group and in the saline group. After reinfusion and in late shock, vessel diameters remained constricted. MAP increased to control levels (106 +/- 5 and 101 +/- 4 mmHg, respectively) following re-infusion in both groups but in late shock it decreased until death in the nontolerant group and decreased only minimally (96 +/- 4 mmHg) in the ENDT-T group. The nontolerant group NE ED50 increased from pre-hemorrhage to late shock (p < .05). The ENDT-T group ED50 was unchanged. The bleeding volumes of the two groups were not different. The survival time of the nontolerant group was 234 +/- 36 min, whereas the ENDT-T group all survived and were sacrificed at 427 +/- 30 min. The response to endothelium-dependent ACH vasodilation in late shock was significantly reduced in the saline group but was unchanged in the ENDT-T group. Alpha 1 receptor activity was maintained in both groups. Alpha 2 receptor activity was attenuated pre-hemorrhage and at 240 min post-hemorrhage in ENDT-T rats. In late shock, alpha 2 receptor activity was attenuated in nontolerant rats. The development of endotoxin tolerance prevents the loss of arteriolar responsiveness to NE and ACh. ENDT-T rats have attenuated alpha 2 receptor activity but not alpha 1 receptor activity.

Macro- and microcirculatory effects of IL-15.

The recently isolated and cloned cytokine interleukin (IL)-15 was studied to ascertain its systemic and local cardiovascular effects. IL-15 was studied in rats anesthetized with 50 mg/kg sodium pentobarbital. In Group I, 10-50 micrograms of IL-15 were administered intravenously. Heart rate and arterial pressure decreased significantly for the duration of the study. In Group II, IL-15 was administered topically to the exposed cremaster muscle in four doses of 10 ng each. Heart rate and arterial pressure decreased significantly for the duration of the study. A1 and A2 arteriolar diameters decreased, but A3 diameters remained unchanged as determined by videomicroscopy. Vasodilation induced by either topical acetylcholine or nitroprusside was abolished by IL-15. Reduced heart rate and arterial pressure suggest reduced cardiac output. Arteriolar constriction due to abolished smooth muscle dilation was caused by IL-15.

Molecular cloning of the human gene encoding lanosterol synthase from a liver cDNA library.

Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] catalyzes the cyclization of (S)-2,3-oxidosqualene to lanosterol in the reaction that forms the sterol nucleus. We report herein the cloning and characterization of the human gene (OSC) encoding lanosterol synthase, a predicted 83 kDa protein of 732 amino acids. The deduced amino acid sequence is 36-40% identical to known yeast and plant homologues and 83% identical to Rattus norvegicus lanosterol synthase. The new gene was shown to encode lanosterol synthase. The yeast lanosterol synthase deficient mutant SMY8 was complemented by the human gene, and a cell-free homogenate of SMY8 expressing the human gene was shown to convert 2,3-oxidosqualene to lanosterol.

Maintenance of dynamic microvascular function and structure in a rat model of endotoxic shock by blockade of the interleukin-1 receptor.

The microvascular and macrovascular effects of IL-1 receptor antagonist (IL-1ra) were examined in rat cremaster muscle A1, A2, and A3 arterioles by videomicroscopy to better define its protective effects during endotoxemia. Mean arterial pressure (MAP), arteriolar diameters, and responses to norepinephrine (NE) and acetylcholine (ACh) were examined hourly after the administration of Escherichia coli endotoxin (6 mg/kg intravenously). Animals received saline (Control) or IL-1ra (.2 mg/kg/min intravenously) beginning 1 h prior to endotoxin. Serum tumor necrosis factor-alpha (TNF-alpha) and nitrate/nitrite (NO) were determined terminally. Aortic endothelium was examined by electron microscopy (EM). All Control animals, but no IL-1ra animals, died within 6 h (p < .01).IL-1ra significantly attenuated endotoxin-induced vasoconstriction of A1 and A2 arterioles (p < .01), while MAP and NE threshold remained at baseline (p < .01 vs. Control). Serum TNF and NO were elevated following endotoxin (p < .001), but only TNF was decreased (p < .005) in animals receiving IL-1ra. Aortic endothelium was damaged in all Control animals but was spared with IL-1 antagonism. IL-1ra increases survival during endotoxic shock and attenuates production of TNF but not NO. IL-1ra maintains MAP, arteriolar diameters, reactivity of arterioles to NE and ACh, and the integrity of the aortic endothelium.

Attenuation of arteriolar alpha 2-adrenoceptor sensitivity during endotoxemia.

It is well documented that adrenergic responses after endotoxin (ENDT) administration are greatly reduced. The hypothesis of this study is that either alpha 1- or alpha 2-receptor activity is attenuated and the other receptor type is minimally affected during ENDT shock. Reactivity of the arterioles of left cremaster muscles of male Wistar rats anesthetized with pentobarbital sodium was studied using videomicroscopy. Femoral mean arterial pressure and first-, second-, third-, and fourth-order arteriolar diameters were measured. In group I, the decreases in arteriolar diameter and half-maximal effective dose (ED50) values with increasing phenylephrine concentration (alpha 1-adrenergic receptor agonist) were similar in all four branching orders before and after ENDT. In group II, the decreases in arteriolar diameter with increasing clonidine concentrations (alpha 2-adrenergic receptor agonist) were effectively attenuated by ENDT, and ED50 values were increased above control in all four branching orders. In group III, idazoxan (alpha 2-receptor antagonist) effectively blocked the vasoconstrictor effects of clonidine but did not affect the responses to phenylephrine before or after ENDT in all four arteriolar orders. In group IV, prazosin (alpha 1-adrenergic receptor antagonist) blocked the vasoconstrictor effects of phenylephrine before and after the administration of ENDT. However, vasoconstriction due to clonidine post-ENDT even at maximal dosage (10(-3) M), was greatly attenuated in all four branching orders as in group II. It is concluded that during endotoxemia the reduced adrenergic vasoconstrictor response of cremaster muscle arterioles is the result of attenuated activity of alpha 2-adrenergic receptors with minimal if any effects on alpha 1-adrenergic receptor activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular endothelium in sepsis and endotoxemia.

Since septic or endotoxin shock was high mortality and morbidity, mechanisms for cardiovascular collapse have been intensely investigated. The vascular response to catecholamines and other agonists is greatly attenuated. The mechanisms are considered to be related to changes in endothelium function. The endothelium releases vasoconstrictor and vasodilator compounds. These autocoids normally interact with systemic and other local vascular controls, but during endotoxemia this balance is severely altered. Large artery endothelia are destroyed whereas the microvessels remain intact and functional. The release of nitric oxide, prostacyclin and endothelin is greatly enhanced. The cytokines from endotoxin seem, however, to be the major causal agents of the syndrome and affect the endothelial or receptors on the endothelia differentially, dependent on tissue location.

Modification of vasopressin microvascular responses by endotoxin, endothelin, and nitric oxide.

The sensitivity of rat cremaster muscle arterioles to topically applied arginine vasopressin (AVP) is greatly increased by endotoxin (ENDT) [1]. The hypothesis is that the increase in vasoconstrictor sensitivity is in part due to modification of the AVP responses by endothelial compounds such as nitric oxide (NO) and endothelin. Reactivity of left cremaster muscle microvessels of pentobarbital anesthetized Sprague-Dawley rats was measured using videomicroscopy. Femoral arterial pressure as well as second and third order arteriolar (A2 and A3) vasoconstrictor threshold responses were determined for topical AVP (10(-15)-10(-6) M). These measurements were repeated in the presence of ENDT (6 mg/kg) alone and in the presence of the NO synthase inhibitor L-NAME (N omega-nitro-L-arginine methyl ester; 1 mg/kg) and ENDT (group 1). The control threshold (M)(-log) for arteriolar constriction by AVP was 9.4 +/- 0.7. After ENDT the threshold decreased significantly (P < 0.05) to 13.8 +/- 0.5, but returned to 9.0 +/- 0.5 after i.v. injected L-NAME. Acetylcholine (ACh) injected i.a. during AVP constriction significantly increased diameters at control and after ENDT, but not after L-NAME. In group 2 the AVP threshold was determined at control, after L-NAME plus hydroquinone (HQ), and at 30, 90, and 120 min post-ENDT in the presence of L-NAME + HQ. The AVP threshold at control was 9.0 +/- 0.3, after L-NAME 9.0 +/- 0.6, and after HQ 8.0 +/- 0.7. After L-NAME + HQ, the threshold was significantly increased to 7.3 +/- 0.2. After ENDT, in the presence of both antagonists, the threshold remained elevated at 7.4 +/- 0.2.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro femoral arterial responses to vasoconstrictor and vasodilator agents in endotoxin shock.

The hypothesis for this study is that the decreased arterial response to catecholamines may be due to the effect of endotoxemia on vessel tone. One control ring was taken from one femoral artery of a Wistar rat and after endotoxin (ENDT) infusion (i.v. 6 mg/kg-1 hr.), one ring was removed from the contralateral artery. The post-ENDT rings were tested in four groups which were determined by the mean arterial pressure (MAP) levels at the time of dissection: 100 mmHg (120 min), 80 mmHg (270 min), 60 mmHg (300 min) or 40 mmHg (330 min). KCl, phenylephrine (PHE) and arginine-vasopressin (AVP) dose-response curves (DR) were obtained at a preload of 500 mg which allowed the maximum response in control rings. When compared at 500 mg preload the maximal active response to all agonists post-ENDT was decreased by about 50%. By increasing the preload on the ENDT rings to 800 mg, the active tension became 2.49 times the active tension of the control rings. Length-tension experiments also showed a greater response for post-ENDT rings and a greater preload at maximum response but the ring circumference was the same. In contrast the in vivo femoral artery diameters at 90 min post-ENDT (100 mmHg) were 82.6% of control. Endothelium-dependent relaxation by acetylcholine (ACh) was abolished by ENDT but endothelium-independent relaxation to nitroprusside (NP) was not affected. It is concluded that the resting tone and active tension of femoral artery smooth muscle is increased by ENDT and the decreased in vivo responsiveness to vasoconstrictor agonists may be the result of vessel constriction due to loss of endothelium. The results also suggest that in vitro comparison of vessels in studies of endotoxin shock be done at the same muscle length rather than at the same preload.