The role of leukocyte function-associated antigen-1 in animal models of inflammation.

Both preclinical and clinical data have identified leukocyte function-associated antigen-1 (LFA-1) as an important component of inflammatory disease states. We evaluated small molecule inhibitors of this glycoprotein in several animal models in which the inflammatory process is dependent on human or non-human primate LFA-1. (R)-5(4-bromobenzyl)-3(3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione, BIRT 377, effectively suppressed the production of human immunoglobulin (IgG) following reconstitution of severe combined immunodeficient (SCID) mice with human peripheral blood mononuclear cells. The BIRT 377 analog, BIX 642, inhibited the cellular infiltrate and increase in skin thickness associated with the delayed-type hypersensitivity reaction in previously immunized squirrel monkeys challenged with antigen. BIX 642 also inhibited the trans-vivo delayed-type hypersensitivity response in the footpads of SCID mice injected with human peripheral blood mononuclear cells and donor-sensitive antigen. These results demonstrate the efficacy of small molecule inhibitors of LFA-1 in preclinical models of inflammation dependent on human or non-human primate LFA-1.

Repeated measurement of intestinal permeability as an assessment of colitis severity in HLA-B27 transgenic rats.

We report on the development of a method for repeated monitoring of mucosal permeability that allows assessment of the severity of colitis and evaluation of treatment efficacy in HLA-B27 transgenic rats. We determined the extent to which intestinal permeability related to stool condition, colon weight, and histological pathology in precolitic and diseased rats up to 29 weeks old. Intestinal permeability was measured by the urinary excretion of iodixanol at 24 h after oral administration. Mean permeability values increased significantly with age in HLA-B27 rats but remained decreased in the background strain Fischer-344 (F-344) control animals. Macroscopic evaluation of HLA-B27 rat colons between 20 and 24 weeks old showed colonic thickening with colonic wet weights increased from 3.4+/-0.13 mg/kg b.wt. in F-344 rats to 6.79+/-0.73 mg/kg b.wt. (p<.05) in HLA-B27 rats. Histological examination of HLA-B27 rat colons confirmed the colonic inflammation as a chronic active mononuclear cell infiltrate. The increase in colon weight was associated with an increase in permeability: 1.16+/-0.17 mg iodixanol versus 5.37+/-1.3 mg of iodixanol in F-344 and HLA-B27 rats, respectively. Three weeks treatment of HLA-B27 rats with cyclosporin A, but not sulfasalazine, showed a dose-dependent decrease in mucosal permeability and colon weight. Neither treatment improved stool condition. We conclude that the measurement of intestinal permeability by iodixanol excretion is a useful biochemical marker that is associated with increases in colonic weight and histological evaluation of inflammation. These data indicate that this technique may be valuable for diagnostic and evaluation purposes in preclinical models of inflammatory bowel disease.

Discovery of non-peptidic P2-P3 butanediamide renin inhibitors with high oral efficacy.

A new series of non-peptidic renin inhibitors having a 2-substituted butanediamide moiety at the P2 and P3 positions has been identified. The optimized inhibitors have IC50 values of 0.8 to 1.4 nM and 2.5 to 7.6 nM in plasma renin assays at pH 6.0 and 7.4, respectively. When evaluated in the normotensive cynomolgus monkey model, two of the most potent inhibitors were orally active at a dose as low as 3 mg/kg. These potent renin inhibitors are characterized by oral bioavailabilities of 40 and 89% in the cynomolgus monkey. Inhibitor 3z (BILA 2157 BS) was selected as candidate for pre-development.

Characterization of the polymorphonuclear leukocyte-induced vasoconstriction in isolated human umbilical veins.

We investigated the contractile effects of both activated and unactivated polymorphonuclear leukocytes (PMNs) on human vascular tissue to characterize the influence of human PMNs on vascular tone. PMNs were added either unactivated or after f-met-leu-phe (fMLP) activation (10(-8) M), into tissue chambers containing human umbilical vein segments under either control or cytokine-treated conditions. The activation state of different PMN preparations was measured by immunofluorescence staining of the adhesion glycoproteins Mac-1 and L-selectin. Both unactivated and activated PMNs induced a cell number-dependent (1.5 x 10(5) to 2 x 10(6) cells/ml) vasoconstriction in human umbilical vein segments. This PMN-induced response was not inhibited by treatment with indomethacin (10(-5) M), superoxide dismutase (2 x 10(-7) M) or L-nitro-monomethyl arginine (10(-4) M). However, treatment of PMNs with the leukotriene biosynthesis inhibitor BIRM-270 partially inhibited (-61 +/- 19%, P <.05) the contraction induced only by unactivated PMNs. Moreover, the supernatant from unactivated, but not that from activated, PMNs elicited a contractile response comparable to that from the addition of cells. We observed a significant correlation between the Mac-1/L-selectin ratio of activated PMNs and the contractile response they generated (r = 0.77, P <.05). The activated PMN response had an endothelium-dependent component, whereas the unactivated PMN response was endothelium-independent. These results suggest that human PMNs of varying activation states have the capacity to modulate vascular smooth muscle tone via distinct mechanisms. Unactivated PMNs appear to modulate tone via a secreted product, whereas the more activated phenotype modulates vascular tone via a cognate interaction with the endothelium.

Captopril-induced hypotension is inhibited by the bradykinin blocker HOE-140 in Na(+)-depleted marmosets.

Inhibition of angiotensin-converting enzyme (ACE) inhibits formation of angiotensin II and, by inhibition of kinin metabolism, may also increase vascular bradykinin. The present experiments were done in sodium-depleted, conscious, unrestrained marmosets (n = 5-11) to examine the contribution of bradykinin to ACE inhibitor-induced hypotension. Aortic blood pressure and heart rate (HR) were monitored via telemetry. After sodium depletion (low-sodium diet and furosemide), captopril (1 mg/kg po) caused a significant (P < 0.05) decrease in mean arterial blood pressure (MABP) (-34 +/- 3 mmHg, maximally, from 79 +/- 2 mmHg) but no change in HR compared with vehicle treatment. The bradykinin receptor antagonist HOE-140 (1 mg/kg sc) significantly inhibited the hypotensive response to captopril and caused marked tachycardia (+133 +/- 14 beats/min from 214 +/- 8 beats/min). HOE-140 (1 mg/kg sc) followed by vehicle administration had no effect on MABP but increased HR similarly. The hypotensive response to captopril was inhibited by HOE-140 regardless of the order of administration or the route of captopril administration (by mouth vs. subcutaneously). The hypotensive response to a renin inhibitor, A-72517 (3 mg/kg sc), was not inhibited by prior HOE-140 administration despite a similar HOE-140-induced tachycardia. These data suggest that the hypotensive effect of captopril in sodium-depleted, conscious marmosets is dependent on functional bradykinin B2 receptors. Also, blockade of B2 receptors uncovers marked tachycardia in this model, suggesting a tonic effect of bradykinin on control of HR in marmosets.

Fractures of the proximal part of the femur.

The orthopaedic surgeon has a multitude of internal fixation devices and techniques available for use in the treatment of subtrochanteric fractures of the proximal femur. The successful use of second-generation locking nails is technically demanding. Close attention to positioning of the patient, reduction of the fracture, placement of the guide-wire, and insertion of the nail and of the proximal and distal locking screws is mandatory. The newer, high-strength hip-screws allow good fixation of a fracture that extends into the piriformis fossa. If medial comminution is present, this technique is best performed in conjunction with indirect reduction and bone-grafting. With proper technique, these devices allow the surgeon to manage predictably a complex subtrochanteric fracture that previously had to be treated with traction or extensive dissection and with (frequently inadequate) internal fixation.

Differential effects of L-NAME on blood pressure and heart rate responses to acetylcholine and bradykinin in cynomolgus primates.

NG-nitro-L-arginine methyl ester (L-NAME) has been reported to have variable effects on the vasodilator response to acetylcholine (ACh) and bradykinin (BK) in vivo. Whether administration of L-NAME affects mean arterial pressure (MAP) or heart rate (HR) responses to ACh or BK was examined in conscious cynomolgus primates. ACh (0.1-10 micrograms/kg i.v.) lowered MAP by 6% to 37%, responses which were inhibited (25-62%) in the presence of L-NAME (1-100 mg/kg i.v.). Although L-NAME increased MAP similarly at doses of 10 and 100 mg/kg, only the 100-mg/kg dose inhibited the hypotensive responses induced by the higher doses of ACh. By comparison, nitroprusside (5 micrograms/kg i.v.)-induced hypotensive responses were not inhibited by L-NAME. Phenylephrine (20 micrograms kg-1 min-1 i.v.) increased MAP and lowered HR to levels statistically similar to that of L-NAME but did not alter ACh-induced hypotensive responses. ACh dose-dependently decreased HR, both in the absence and presence of L-NAME or phenylephrine. In pentobarbital-anesthetized monkeys, ACh-induced hypotensive responses were inhibited by 75% to 94% in the presence of L-NAME; BK (0.3-1 microgram/kg i.v.) responses were only modestly affected (< or = 50%). Therefore, in conscious primates, L-NAME affects the basal release of nitric oxide (NO) at lower doses than those required to inhibit its release stimulated by ACh. Also, L-NAME does not appear to act as a cholinergic antagonist or affect the functional mechanisms that control baroreflex responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Loss of heterozygosity in sporadic human breast carcinoma: a common region between 11q22 and 11q23.3.

The development of sporadic human breast cancer is associated with the accumulation of genetic alterations on several chromosomes. In the case of chromosome 11, loss of heterozygosity (LOH) at loci on the short arm has been well documented and suggests the presence of a suppressor gene(s) at 11p15.5. However, the evidence for similar events on the long arm is less compelling. Here, we determined the prevalence of LOH for chromosome 11q in 44 malignant and 3 benign cases of unselected sporadic breast tumor samples. We found that alteration of chromosome 11q is common in the pathogenesis of breast cancer as 19 of 44 (43%) malignant tumor specimens exhibited LOH. Eleven (58%) of these genetic alterations were specific to the long arm of the chromosome. The smallest region of shared LOH places the target between 11q22 and 11q23.3, the same general region frequently altered in cancers of the ovary, colon, skin, and uterine cervix, perhaps indicating the location of a tumor suppressor gene or genes of importance in each of these different tumor types.

Sodium depletion in conscious cynomolgus monkeys attenuates baroreflex sensitivity independently of prostaglandins.

We tested the hypothesis that baroreflex attenuation during sodium depletion is due to increased prostaglandin (PG) levels. We studied baroreflex sensitivity before and after PG synthesis inhibition in conscious cynomolgus monkeys. Arterial pressure and pulse interval (PI) were measured during intravenous infusions of phenylephrine (1-20 micrograms.kg-1.min-1, n = 6) and nitroprusside (1-10 micrograms.kg-1.min-1, n = 7). Infusions were repeated 30 min after indomethacin (Indo, 6 mg/kg iv). The slope (in ms/mmHg) of the mean arterial blood pressure-PI plot was used as an index of baroreflex sensitivity. Plasma renin activity (PRA) was elevated (47.9 +/- 9.7 vs. 8.8 +/- 3.3 ng angiotensin I.ml-1.h-1) after sodium depletion (P < 0.05). Baroreflex sensitivity to hypotension and hypertension was significantly (P < 0.05) attenuated by sodium depletion (3.69 +/- 0.9 vs. 0.9 +/- 0.1 ms/mmHg and 7.38 +/- 0.6 vs. 5.04 +/- 0.9 ms/mmHg, respectively). Indo decreased PRA to 28.6 +/- 5.7 ng angiotensin I.ml-1.h-1 (P < 0.05) in sodium-depleted monkeys and decreased heart rate -21 +/- 3.7 from a baseline of 166 +/- 9.40 beats/min in normal monkeys and -22 +/- 2.9 from a baseline of 191 +/- 7.9 beats/min in low-sodium monkeys (P < 0.05). Indo did not significantly change baroreflex sensitivity in either group. Thus the baroreflex was attenuated in conscious nonhuman primates during sodium depletion; acute PG synthesis blockade did not improve baroreflex sensitivity. Indo decreased heart rate without changing arterial pressure; suggesting that PGs caused a downward resetting of the pressure-heart rate relationship.

Effects of losartan (DuP753) and enalaprilat on the mean arterial pressure response to phenylephrine.

BACKGROUND: We have found that low-dose infusion of angiotensin II (Ang II) selectively potentiates the mean arterial pressure (MAP) response to phenylephrine in pentobarbital-anesthetized rabbits. OBJECTIVE: To examine whether endogenous Ang II levels in normotensive rabbits maintained on a normal-salt diet exert a potentiating effect on the MAP response to phenylephrine. METHODS: We compared the effects of enalaprilat (0.3 mg/kg per min for 5 min bolus, 1 mg/kg per h infusion), losartan (DuP753; 4 mg/kg bolus, 2 mg/kg per h infusion) and vehicle administration on the MAP response to infusions of phenylephrine that were increased incrementally (2.5, 5 and 10 micrograms/kg per min). RESULTS: Enalaprilat decreased MAP significantly, whereas no maintained change was observed with losartan or vehicle. Phenylephrine infusions elevated MAP significantly and dose-dependently in all of the rabbits studied, but this effect was attenuated significantly in the rabbits given losartan compared with in those given vehicle or enalaprilat. The heart rate responses were not significantly different among the three groups. CONCLUSION: We conclude that inhibition of the renin-angiotensin system at two distinct sites results in different MAP responses to phenylephrine infusion.

Vasoconstrictor and vasodilator effects of serotonin in the isolated rabbit kidney.

The renal vascular effects of serotonin (5-HT) in vivo vary among preparations, which may reflect that this autacoid can modulate the vascular response to certain spasmogens. We investigated this phenomenon in the isolated rabbit kidney perfused under constant flow (5 ml/min) with Krebs-bicarbonate buffer. Dose-response experiments to 5-HT were conducted before and during an infusion of half-maximal effective doses of norepinephrine (NE), phenylephrine (PE) or endothelin-1 (ET-1). Each infusion was varied to raise the perfusion pressure (PP) from a baseline of 18-28 mm Hg to a level of 80-100 mm Hg. In the absence of infused NE, PE or ET-1, bolus injections of 5-HT had little or no effect. However, in the presence of NE, 5-HT injections of 20 pmol to 20 nmol caused a dose-dependent increase in PP; higher doses (0.4-4 mumol) of 5-HT caused a dose-dependent decrease in PP. Similar results with 5-HT were obtained in the presence of an infusion of PE. However, in the presence of ET-1, 5-HT injections elicited only a modest increase in PP, which was significantly less than the increase in the presence of NE; and they did not have the vasodilator effect on the isolated perfused kidney. Histamine injections only increased but did not decrease the PP in PE-precontracted renal vasculature. Infusion of 5-HT at 20 nmol/min potentiated the dose-dependent effect of NE on PP, but 5-HT infusion of 1 mumol/min attenuated the effect of NE on PP.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional differences in the effects of septic shock on vascular reactivity in the rabbit.

Experiments were performed to investigate the vascular reactivity in both large and resistance artery preparations from an animal model of septic shock. New Zealand White rabbits were injected with a priming dose of Escherichia coli lipopolysaccharide (LPS), 15 micrograms/kg i.v., 18 hr before an i.v. dose of 200 to 2000 micrograms/kg under pentobarbital anesthesia. The second LPS challenge dropped mean blood pressure from 79 +/- 4 to 27 +/- 5 mm Hg in approximately 1 hr. At this time animals were sacrificed with the central ear arteries and kidneys being isolated for alignment in a Krebs-bicarbonate buffer perfusion apparatus to monitor perfusion pressure under constant flow conditions. Individual dose-response curves (DRCs) for norepinephrine (NE) and histamine were performed to assess contractile function. For examining vascular relaxant function, DRCs for methacholine (MC) and nitroprusside (NP) were conducted during a submaximal infusion of NE. The DRCs to NE and histamine were shifted to the right by 2- and 2.7-fold, respectively, in isolated ear artery preparations from LPS-treated vs. vehicle-treated animals. There was no difference in contractile function (using NE) in the two groups of perfused kidneys. The relaxation DRCs to MC were similar in the ear artery preparations from the two treatment groups whereas, in isolated kidneys, the relaxation to MC was significantly attenuated, by an average of 26 +/- 2% at each of six doses, in preparations from LPS-treated animals. The relaxation to NP was similar between the LPS- and vehicle-treated animals in the ear artery and kidney preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride analogs inhibit L-type calcium channels and display calcium entry blocker activity.

Three structural classes of commonly used amiloride analogs, molecules derivatized at the terminal guanidino-nitrogen, the five-position pyrazinoyl-nitrogen, or di-substituted at both of these positions, inhibit binding of the L-type Ca2+ channel modulators diltiazem, gallopamil, and nitrendipine to porcine cardiac sarcolemmal membrane vesicles. The rank order of inhibitory potencies among the various derivatives tested is well defined with amiloride being the least potent. Saturation binding studies indicate that inhibition of ligand binding results primarily from effects on Kd. Ligand dissociation measurements suggest that amiloride derivatives do not associate directly at any of the known sites in the Ca2+ entry blocker receptor complex. In addition, these compounds do not compete at the "Ca2+ coordination site" within the channel. However, studies with inorganic and substituted diphenylbutylpiperidine Ca2+ entry blockers reveal that amiloride analogs interact at a site on the channel where metal ions bind and occlude the pore. Photolysis experiments performed with amiloride photoaffinity reagents confirm that a specific interaction occurs between such probes and the channel protein. Upon photolysis, these agents produce concentration- and time-dependent irreversible inactivation of Ca2+ entry blocker binding activities, which can be protected against by either verapamil or diltiazem. 45Ca2+ flux and voltage-clamp experiments performed with GH3 anterior pituitary cells demonstrate that amiloride-like compounds inhibit L-type Ca2+ channels directly. Moreover, these compounds block contraction of isolated vascular tissue in pharmacological assays. Electrophysiological experiments indicate that they also inhibit T-type Ca2+ channels in GH3 cells. Taken together, these results demonstrate unequivocally that amiloride analogs display significant Ca2+ entry blocker activity in both ligand binding and functional assays. This property, therefore, can seriously complicate the interpretation of many in vitro and in vivo studies where amiloride analogs are used to elicit inhibition of other transport systems (e.g. Na-Ca and Na-H exchange).

Acute hypotensive responses to peptide inhibitors of renin in conscious monkeys: an effect on blood pressure independent of plasma renin inhibition.

In order to investigate the hypotensive mechanisms of action of peptide renin inhibitors, blood pressure responses to five renin inhibitors were compared with those to the angiotensin converting enzyme inhibitor, enalaprilat, in conscious African green and rhesus monkeys. (3S-4S)-4-amino-5-cyclohexyl-3-hydroxy pentanoic acid (ACHPA)-containing renin inhibitory peptide (ACRIP) and enalaprilat both decreased blood pressure in euvolemic and volume-depleted African green monkeys. However, while a maximum dose of enalaprilat reduced blood pressure to 80 +/- 4 and 56 +/- 4 mmHg in the euvolemic and volume-depleted monkeys, respectively, ACRIP lowered pressure to life-threatening levels (less than 40 mmHg) under both conditions. The relative potencies of ACRIP and four other renin inhibitors for inhibiting in vitro plasma renin activity (PRA; IC50) were compared with their potencies in reducing blood pressure by 15 mmHg (ED15 mmHg) and lowering blood pressure more than enalaprilat in volume-depleted rhesus monkeys. All renin inhibitors lowered blood pressure significantly beyond the maximal response to enalaprilat. Despite a significant correlation (r = 0.99, P less than 0.05) between the in vitro PRA inhibitory potency and the in vivo ED15 mmHg, doses which lowered blood pressure beyond the maximal responses to enalaprilat were not significantly correlated (r = 0.53, P greater than 0.05) with the in vitro PRA IC50 values. Furthermore, the profound depressor responses to renin inhibitors in rhesus monkeys were accompanied by increases in the heart rate and decreases in pulse pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of atrial natriuretic factor-induced vasodilation.

ANF can potentially elicit vasorelaxation in vitro which is typically associated with an elevation in tissue levels of cGMP. Hypotension with vasodilation can be observed upon injection of ANF in vivo, however, infusion of the peptide often results in a decreased blood pressure due to a fall in cardiac output, This apparent discrepancy may reflect some of the distinguishing characteristics of ANF-induced vasorelaxation which include activation of particulate guanylate cyclase, a marked regional vascular selectivity, species differences in the relaxation profile and a variable sensitivity depending on the type and degree of contractile preload.

Enhanced release of atrial natriuretic factor by endothelin in atria from hypertensive rats.

Intravenous (bolus) administration of endothelin results in a transient fall in blood pressure that is accentuated in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto normotensive rats (WKY). In attempting to discern possible mechanisms underlying this depressor response, we examined the ability of endothelin to release atrial natriuretic factor (ANF) from isolated, spontaneously contracting atria from SHR and WKY. Isolated right atria were suspended under 3.0 g of resting force in tissue baths with the amount of immunoreactive ANF (irANF) released after exposure to endothelin assessed by radioimmunoassay. Endothelin (10(-8) and 10(-7) M) caused a concentration-dependent increase (1.5-4.5-fold) in the release of irANF, which was significantly greater in atria of SHR compared with WKY. The greater release of irANF in atria of SHR versus WKY was not related to tissue weight or changes in contractile rate or force induced by endothelin. Therefore, endothelin appears to cause a direct release of irANF from rat right atria in vitro. As found for the depressor response in vivo, endothelin is more efficacious in the hypertensive compared with the normotensive atrial preparation. Release of ANF may be important in the hypotensive response to endothelin in vivo.

Prominent depressor response to endothelin in spontaneously hypertensive rats.

Administration of endothelin (0.03-3.0 micrograms/kg i.v.) caused transient depressor responses followed by sustained pressor responses in anesthetized spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The initial depressor response occurred at lower doses (0.1 versus 0.3 micrograms/kg i.v.) in SHR versus WKY. The secondary pressor response was attenuated in SHR compared to WKY in both the threshold dose (3.0 versus 0.1 microgram/kg i.v.) and maximum effect at high doses (52 versus 91% at 3.0 micrograms/kg i.v.). In conscious SHR and WKY, endothelin elicited comparable initial depressor responses with increases in heart rate; the secondary pressor responses were attenuated compared to those in anesthetized rats. Therefore endothelin elicits a prominent depressor response, which may be associated with afterload reduction, in SHR.

Glyburide blocks the relaxation response to BRL 34915 (cromakalim), minoxidil sulfate and diazoxide in vascular smooth muscle.

BRL 34915 [6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl) 2H-benzo(b) pyran-3-ol], minoxidil sulfate and diazoxide may relax vascular smooth muscle via hyperpolarization due to an opening of membrane potassium channels. We therefore examined the effects of several potassium channel antagonists on the relaxation response to these vasodilators in isolated rat portal venous strips which were mounted in vitro for detecting changes in isometric force. BRL 34915 (IC50 = 4.7 X 10(-8) M), minoxidil sulfate (IC50 = 1.4 X 10(-7) M) and diazoxide (IC50 = 5 X 10(-6) M) elicited concentration-dependent relaxations of the spontaneous, myogenic contractions in venous strips. The relatively nonselective potassium channel antagonists tetraethylammonium ion (0.3-10 X 10(-3) M) and 4-aminopyridine (1-10 X 10(-3) M) caused concentration-dependent shifts (5- to 50-fold) in the relaxation responses to each vasodilator. Charybdotoxin (up to 10(-7) M) and apamin (up to 10(-7) M), known to be antagonists of high and low conductance calcium-activated potassium channels, respectively, had no inhibitory effect on the relaxation-response curves to BRL 34915, minoxidil sulfate or diazoxide. Glyburide (10(-7) to 3 X 10(-5) M), a sulfonylurea which has been shown to block the ATP-modulated potassium channel in insulin-secreting cells, caused concentration-dependent shifts to the right (up to 100-fold) of the IC50 value for BRL 34915 and diazoxide, and at 10(-6) M, abolished the relaxation response to minoxidil sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)