Riding out the storm: depleted fat stores and elevated hematocrit in a small bodied endotherm exposed to severe weather.

In the mid-continental grasslands of North America, climate change is increasing the intensity and frequency of extreme weather events. Increasingly severe storms and prolonged periods of elevated temperatures can impose challenges that adversely affect an individual's condition and, ultimately, survival. However, despite mounting evidence that extreme weather events, such as heavy rain storms, can impose short-term physiological challenges, we know little regarding the putative costs of such weather events. To determine the consequences of extreme weather for small endotherms, we tested predictions of the relationships between both severe precipitation events and wet bulb temperatures (an index that combines temperature and humidity) prior to capture with body composition and hematocrit of grasshopper sparrows (Ammodramus savannarum) caught during the breeding season at the Konza Prairie Biological Station, Kansas, USA, between 2014 and 2016. We measured each individual's fat mass, lean mass and total body water using quantitative magnetic resonance in addition to their hematocrit. Individuals exposed to storms in the 24 hours prior to capture had less fat reserves, more lean mass, more water and higher hematocrit than those exposed to moderate weather conditions. Furthermore, individuals stored more fat if they experienced high wet bulb temperatures in the week prior to capture. Overall, the analysis of these data indicate that extreme weather events take a physiological toll on small endotherms, and individuals may be forced to deplete fat stores and increase erythropoiesis to meet the physiological demands associated with surviving a storm. Elucidating the potential strategies used to cope with severe weather may enable us to understand the energetic consequences of increasingly severe weather in a changing world.

Social interactions do not drive territory aggregation in a grassland songbird.

Understanding the drivers of animal distributions is a fundamental goal of ecology and informs habitat management. The costs and benefits of colonial aggregations in animals are well established, but the factors leading to aggregation in territorial animals remain unclear. Territorial animals might aggregate to facilitate social behavior such as (1) group defense from predators and/or parasites, (2) cooperative care of offspring, (3) extra-pair mating, and/or (4) mitigating costs of extra-pair mating through kin selection. Using experimental and observational methods, we tested predictions of all four hypotheses in a tallgrass prairie in northeast Kansas, United States. Grasshopper Sparrow (Ammodramus savannarum) males formed clumps of territories in some parts of the site while leaving other apparently suitable areas unoccupied. Despite substantial sampling effort (653 territories and 223 nests), we found no support for any hypothesized social driver of aggregation, nor evidence that aggregation increases nest success. Our results run counter to previous evidence that conspecific interactions shape territory distributions. These results suggest one of the following alternatives: (1) the benefits of aggregation accrue to different life-history stages, or (2) the benefits of territory aggregation may be too small to detect in short-term studies and/or the consequences of aggregation are sufficiently temporally and spatially variable that they do not always appear to be locally adaptive, perhaps exacerbated by changing landscape contexts and declining population sizes.

[Selective digestive decontamination: the light as changed from red to green]. / La décontamination digestive sélective: le feu est passé du rouge au vert.

OBJECTIVES: To study the efficacy of selective digestive decontamination (SDD) for the prevention of nosocomial infections, particularly pneumonia, as well as its impact on the emergence of multiresistant bacteria. DATA SOURCES: Data collected from the Pubmed: original articles, review articles and editorial published on SDD. The keywords were: selective digestive decontamination, pneumonia, intensive care unit, infection. DATA SELECTION: Ten randomized clinical trials performed since 1995 in mechanically ventilated adult patients hospitalized in intensive care unit. RESULTS: The rationale for the use of SDD consists on the parenteral administration of a short course of antibiotic associated with the topical use of non-absorbable antibiotics directed against Gram negative bacteria. Five randomized studies described a reduction in the incidence of pneumonia associated with SDD. Only one study has showed a decrease in mortality rate. The other five studies, which present some methodological limitations, concluded the lack of efficacy of SDD. Regarding the emergence of multiresistant bacteria, the literature underlines the role of environment. The use of SDD seems to trigger the resistance in endemic areas, while these are softened in the units with a good control of their ecology. CONCLUSION: The data from the literature provide arguments to use SDD in targeted patient populations like multiple traumas in intensive care units, which have a low rate of multiresistant bacteria.

Hemodynamic and metabolic effects of low-dose vasopressin infusions in vasodilatory septic shock.

OBJECTIVE: To investigate the physiologic effects of exogenous vasopressin as a potential alternative to traditional high-dose catecholamine therapy for septic patients with vascular hyporeactivity to catecholamines. DESIGN: Prospective, case-controlled study. SETTING: Intensive care unit of a university hospital. PATIENTS: Vasopressin was infused in 16 critically ill septic patients who remained persistently hypotensive despite infusions of pharmacologic doses of catecholamines. INTERVENTION: Continuous intravenous infusion of vasopressin at 0.04 units/min for 16 hrs, in place of escalating the amount of catecholamines being infused. MEASUREMENTS AND MAIN RESULTS: After administration of vasopressin, systemic vascular resistance and mean arterial pressure were immediately and significantly increased in comparison with the values obtained just before vasopressin. When the vasopressin infusions were discontinued, mean arterial pressure decreased immediately and dramatically. We did not detect any obvious adverse cardiac effects during the vasopressin infusions. Vasopressin had no effect on other hemodynamic parameters or any of the metabolic parameters studied, including measures of oxygenation, plasma glucose, or electrolytes. Urine output increased significantly during the administration of vasopressin, although this effect may be nonspecific. Lactate concentrations decreased, particularly in the survival group, but the decreases were not significant. Overall survival was 56%. CONCLUSIONS: Low-dose vasopressin infusions increased mean arterial pressure, systemic vascular resistance, and urine output in patients with vasodilatory septic shock and hyporesponsiveness to catecholamines. The data indicate that low-dose vasopressin infusions may be useful in treating hypotension in these patients.

A prospective, randomized study comparing percutaneous with surgical tracheostomy in critically ill patients.

OBJECTIVE: To determine the relative cost-effectiveness of percutaneous dilational tracheostomy (PDT) and surgical tracheostomy (ST) in critically ill patients. DESIGN: Prospective randomized study. SETTING: Medical, surgical, and coronary intensive care units at Barnes-Jewish Hospital, a tertiary care medical center. PATIENTS: Eighty critically ill mechanically ventilated patients requiring elective tracheostomy. INTERVENTIONS: Randomization to either PDT performed in the intensive care unit or ST performed in the operating room. MEASUREMENTS AND MAIN RESULTS: Treatment groups were well matched with respect to age (PDT, 65.44 +/- 2.82 [mean +/- se] years; ST, 61.4 +/- 2.89 years, p = Ns), gender (PDT, 45% males; ST, 47.5% males, p = NS), severity of illness (Acute Physiology and Chronic Health Evaluation II score: PDT, 16.87 +/- 0.84; ST, 17.88 +/- 0.92, p = NS), and principle diagnosis. PDT was performed more quickly (PDT, 20.1 +/- 2.0 mins; ST, 41.7 +/- 3.9 mins, p < .0001) and was associated with lower patient charges than ST (total patient charges: PDT, 1,569 dollars +/- 157 dollars vs. ST, 3,172 dollars +/- 114 dollars; equipment/supply charges: PDT, 688 dollars +/- 103 dollars vs. ST, 1,526 dollars +/- 87 dollars; professional charges: PDT, 880 dollars +/- 54 dollars vs. ST, 1,647 dollars +/- 50 dollars; p < .0001 for all). There were no differences in days intubated before tracheostomy (PDT, 12.7 +/- 1.1 days; ST, 15.6 +/- 1.9, p = .20), intensive care unit length of stay (PDT, 24.5 +/- 2.5 days; ST, 28.5 +/- 3.1 days, p = .33), or hospital length of stay (PDT 49.7 +/- 4.2 days; ST, 43.7 +/- 3.5 days, p = .28) when we compared these two techniques. CONCLUSIONS: PDT is a cost-effective alternative to ST. The reduction in patient charges associated with PDT in this study resulted from the procedure being performed in the intensive care unit, thus eliminating the need for operating room facilities and personnel. PDT may become the procedure of choice for electively establishing tracheostomy in the appropriately selected patient who requires long-term mechanical ventilation.

Hyperbaric hyperoxia suppresses growth of Staphylococcus aureus, including methicillin-resistant strains.

PURPOSE: We investigated the effects of increased oxygen tension on the in vitro growth of Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), and Escherichia coli (E. coli). METHODS: The effects of oxygen tension [normobaric normoxia (21% O(2) at 1 atm), normobaric hyperoxia (100% O(2) at 1 atm), hyperbaric normoxia (21% O(2) at 2 atm), and hyperbaric hyperoxia (100% O(2) at 2 atm) on the in vitro growth of MRSA, MSSA, and E. coli were investigated by population analysis. RESULTS: Compared with normobaric normoxia, a 90-min exposure to hyperbaric hyperoxia significantly inhibited growth of both MRSA (by 25.0 +/- 3.0%, mean +/- SEM; P < 0.01) and MSSA (by 24.0 +/- 3.3%; P < 0.01). Normobaric hyperoxia and hyperbaric normoxia were without effect. In contrast, the growth of E. coli was not affected by any of the above treatments. CONCLUSION: Our results indicate that the bacterium S. aureus, including resistant strains, is susceptible to oxygen stress. The observation that relatively brief (90-min) treatment with hyperbaric hyperoxia is sufficient to produce significant growth inhibition suggests that hyperbaric hyperoxia may be useful in the treatment of serious staphylococcal infections.

iNOS gene expression modulates microvascular responsiveness in endotoxin-challenged mice.

Septic shock is characterized by vasodilation and decreased responsiveness to vasoconstrictors. Recent studies suggest this results from nitric oxide (NO) overproduction after expression of the calcium-independent isoform of NO synthase (iNOS) in smooth muscle cells. However, direct evidence linking iNOS (NOS2) expression and decreased microvascular responsiveness after septic stimuli is lacking. In the present study, we determined the effect of bacterial lipopolysaccharide (LPS, 20 mg/kg, IP) on smooth muscle contraction and endothelial relaxation in mesenteric resistance arteries from wild-type and iNOS knockout mice. Four hours after challenge with LPS or saline in vivo, concentration-dependent responses to norepinephrine (NE) and acetylcholine (NE+ACh) were measured in cannulated, pressurized vessels ex vivo. In vessels from wild-type mice, NE-induced contraction was markedly impaired after LPS, and pretreatment with the iNOS inhibitor aminoguanidine (AG) partly restored the NE contraction. In contrast, NE contraction in microvessels from iNOS knockout mice was unaffected by LPS. ACh-induced relaxation was unaffected by LPS in vessels from either genotype. These data provide direct evidence that iNOS gene expression mediates the LPS-induced decrease in microvascular responsiveness to vasoconstrictors. Moreover, the observation that AG did not fully restore NE contraction after LPS, whereas iNOS gene deficiency did, suggests that iNOS expression plays a central role in the development of the NO-independent effect of LPS on microvascular responsiveness. Finally, our data indicate that LPS or iNOS expression has little effect on endothelium-dependent relaxation, and eNOS activity does not appear to play a role in the decreased smooth muscle responsiveness after LPS in this model. The full text of this article is available at http://www.circresaha.org.

Relation between outer and luminal diameter in cannulated arteries.

Resistance in blood vessels is directly related to the inner (luminal) diameter (ID). However, ID can be difficult to measure during physiological experiments because of poor transillumination of thick-walled or tightly constricted vessels. We investigated whether the wall cross-sectional area (WCSA) in cannulated arteries is nearly constant, allowing IDs to be calculated from outer diameters (OD) using a single determination of WCSA. With the use of image analysis, OD and ID were directly measured using either transillumination or a fluorescent marker in the lumen. IDs from a variety of vessel types were calculated from WCSA at several reference pressures. Calculated IDs at all of the reference WCSA were within 5% (mean <1%) of the corresponding measured IDs in all vessel types studied, including vessels from heterozygote elastin knockout animals. This was true over a wide range of transmural pressures, during treatment with agonists, and before and after treatment with KCN. In conclusion, WCSA remains virtually constant in cannulated vessels, allowing accurate determination of ID from OD measurement under a variety of experimental conditions.

Novel arterial pathology in mice and humans hemizygous for elastin.

Obstructive vascular disease is an important health problem in the industrialized world. Through a series of molecular genetic studies, we demonstrated that loss-of-function mutations in one elastin allele cause an inherited obstructive arterial disease, supravalvular aortic stenosis (SVAS). To define the mechanism of elastin's effect, we generated mice hemizygous for the elastin gene (ELN +/-). Although ELN mRNA and protein were reduced by 50% in ELN +/- mice, arterial compliance at physiologic pressures was nearly normal. This discrepancy was explained by a paradoxical increase of 35% in the number of elastic lamellae and smooth muscle in ELN +/- arteries. Examination of humans with ELN hemizygosity revealed a 2. 5-fold increase in elastic lamellae and smooth muscle. Thus, ELN hemizygosity in mice and humans induces a compensatory increase in the number of rings of elastic lamellae and smooth muscle during arterial development. Humans are exquisitely sensitive to reduced ELN expression, developing profound arterial thickening and markedly increased risk of obstructive vascular disease.

Is guanosine-5'-triphosphate involved in calcium-activation of contractile proteins in vascular smooth muscle?

Isometric tension was measured to investigate the effects of guanosine-5'-triphosphate (GTP) on the run-down of myofilament Ca2+ sensitivity in isolated rat mesenteric arteries permeabilized with beta-escin. The Ca2+ sensitivity assessed by the EC50 value for the Ca2+ (0.1-100 microM)-tension relationship progressively runs down in the control strips, while it was well-preserved for 5-successive Ca2+ applications in the presence of GTP (50 microM); no significant difference was found in the Ca2+ sensitivity observed with the 1st Ca2+ application between the control and GTP-treated strips. Guanosine-5'-(2-O-thio) diphosphate (GDP beta S, 100 microM) significantly decreased the Ca2+ sensitivity with the 1st Ca2+ application and eliminated the run-down of Ca2+ sensitivity. GTP (3-150 microM), applied to the strips submaximally precontracted with Ca2+, had a little effect on the Ca2+ contractions in the early stage of experiments, but dramatically enhanced the Ca2+ contractions in their later stage; its latter effect was mimicked by guanosine-5'-(3-O-thio) triphosphate (GTP gamma S) and reversed by GDP beta S (100 microM). The results suggest: 1) loss of endogenous GTP following permeabilization is involved in the run-down of Ca2+ sensitivity; and 2) activation of G-proteins is involved in Ca(2+)-activation of contractile proteins.

Vascular actions of 'caged' phenylephrine analogs depend on the structure and site of attachment of the 2-nitrobenzyl group.

In the experiments presented in this article, the effects of four caged analogs of the alpha 1-adrenergic agonist phenylephrine (PE) on the properties of small (100-200 microns outer diameter), isolated rat mesenteric arteries were compared. The four caged PE analogs contained either an unsubstituted (analogs I and II) or an alpha-carboxy substituted (analogs III and IV) 2-nitrobenzyl group attached to the phenolic oxygen atom (O-linked; analogs II and IV) or to the amino group (N-linked; analogs I and III) of PE. The structure of each caged PE analog was confirmed by UV, IR and 1H NMR spectral analysis. For physiological experiments, photolysis of the caged PE analogs was accomplished with a Hi-Tech Scientific flashlamp, and vascular smooth muscle contraction was measured with a computer-based image analysis system. In some experiments, the fura-2 ratiometric technique was used to examine the effects of the caged PE analogs on intracellular Ca2+ levels. At concentration < or = 10(-6) M, none of the four analogs displayed measurable intrinsic vasoconstricting activity, that is, vasoconstrictions were only observed following light flashes, consistent with the release of free PE. At concentrations > or = 10(-5) M, however, both O-linked compounds (analogs II and IV) and the alpha-carboxy substituted N-linked caged PE (analog III) produced vasoconstriction prior to photolysis. In contrast, no intrinsic vasoconstricting activity was evident with the unsubstituted N-linked caged PE (analog I) at concentrations up to 300 microM (the highest concentration tested). At concentrations > or = 10 microM, the O-linked unsubstituted caged PE (analog II) also had intrinsic vasodilating activity and markedly attenuated vasoconstrictions and increases in intracellular Ca2+ produced by high KCl. Similar effects were observed with the N-linked caged PE analogs (I and III) at > or = 100 microM, whereas no measurable relaxations were seen with the alpha-carboxy O-linked caged PE analog (i.v.) at concentrations up to 300 microM (the highest concentration tested). Taken together, the results presented here demonstrate that the N-linked unsubstituted caged PE analog (I) can be used reliably at concentrations up to 100 microM and is, therefore, the analog of choice for physiological studies of alpha 1-receptor-mediated events.

Volatile anesthetic actions on contractile proteins in membrane-permeabilized small mesenteric arteries.

BACKGROUND: Volatile anesthetics have been shown to have vasodilating or vasoconstricting actions in vitro that may contribute to their cardiovascular effects in vivo. However, the precise mechanisms of these actions in vitro have not been fully elucidated. Moreover, there are no data regarding the mechanisms of volatile anesthetic action on small resistance arteries, which play a critical role in the regulation of blood pressure and blood flow. METHODS: With the use of isometric tension recording methods, volatile anesthetic actions were studied in intact and beta-escin-membrane-permeabilized smooth muscle strips from rat small mesenteric arteries. In experiments with intact muscle, the effects of-halothane (0.25-5.0%), isoflurane (0.25-5.0%), and enflurane (0.25-5.0%) were investigated on high K(+)-induced contractions at 22 degrees C and 35 degrees C. All experiments were performed on endothelium-denuded strips in the presence of 3 microM guanethidine and 0.3 microM tetrodotoxin to minimize the influence of nerve terminal activities. In experiments with membrane-permeabilized muscle, the effects of halothane (0.5-4.0%), isoflurane (0.5-4.0%), and enflurane (0.5-4.0%) on the half-maximal and maximal Ca(2+)-activated contractions were examined at 22 degrees C in the presence of 0.3 microM ionomycin to eliminate intracellular Ca2+ stores. RESULTS: In the high K(+)-stimulated intact muscle, all three anesthetics generated transient contractions, which were followed by sustained vasorelaxation. The IC50 values for this vasorelaxing action of halothane, isoflurane, and enflurane were 0.47 vol% (0.27 mM), 0.66 vol% (0.32 mM), and 0.53 vol% (0.27 mM), respectively, at 22 degrees C and were 3.36 vol% (0.99 mM), 3.07 vol% (0.69 mM), and 3.19 vol% (0.95 mM), respectively, at 35 degrees C. Ryanodine (10 microM) eliminated the anesthetic-induced contractions but had no significant effect on the anesthetic-induced vasorelaxation in the presence of high K+. In addition, no significant differences were observed in the dose dependence of the direct vasodilating action among these anesthetics with or without ryanodine at either the low or the high temperature. However, significant differences were observed in the vasoconstricting actions among the anesthetics, and the order of potency was halothane > enflurane > isoflurane. The Ca(2+)-tension relation in the membrane-permeabilized muscle yielded a half-maximal effective Ca2+ concentration (EC50) of 2.02 microM. Halothane modestly but significantly inhibited 3 microM (approximately the EC50) and 30 microM (maximal) Ca(2+)-induced contractions. Enflurane slightly but significantly inhibited 3 microM but not 30 microM Ca2+ contractions. Isoflurane did not significantly inhibit either 3 microM or 30 microM Ca2+ contractions. CONCLUSIONS: Halothane, isoflurane, and enflurane have both vasoconstricting and vasodilating actions on isolated small splanchnic resistance arteries. The direct vasoconstricting action appears to result from Ca2+ release from the ryanodine-sensitive intracellular Ca2+ store. The vasodilating action of isoflurane in the presence of high K+ appears to be attributable mainly to a decrease in intracellular Ca2+ concentration, possibly resulting from inhibition of voltage-gated Ca2+ channels. In contrast, the vasodilating actions of halothane and enflurane in the presence of high K+ appears to involve inhibition of Ca2+ activation of contractile proteins as well as a decrease in intracellular Ca2+ concentration in smooth muscle.

Brachytherapy-related complications for medically inoperable stage I endometrial carcinoma.

PURPOSE: The current study was conducted to investigate the incidence and risk factors for medical complications associated with low dose rate brachytherapy in patients with medically inoperable Stage I endometrial cancer treated with irradiation alone. METHODS AND MATERIALS: From 1965 through 1991 at Mallinckrodt Institute of Radiology, 150 implants were performed on 96 patients who were deemed medically unfit for hysterectomy because of advanced age, obesity, and various medical problems. The records of these patients were examined retrospectively to determine the incidence of medical complications that occurred in the first 30 days following the initiation of brachytherapy. The association of risk factors that precluded major surgery and the occurrence of brachytherapy-related complications was examined by logistic regression. RESULTS: Of these 96 patients, 40 patients were older than 75 years, and 31 patients were deemed morbidly obese. Medical problems included hypertension in 45 patients, and diabetes in 37; there was a history of congestive heart failure in 23, stroke in 11, myocardial infarction in 10, and thromboembolism in 8. There were concurrent malignancies in five patients. Implants were performed using intrauterine Simon-Heyman capsules, tandems, and vaginal ovoids in all patients. General anesthesia was used for 98 implants, spinal anesthesia for 26, local anesthesia for 25, and epidural anesthesia for 1. The duration of anesthesia ranged from 30 to 120 min (median, 60 min). The duration of radioisotope application ranged from 11 to 96 h (median, 46 h). Preventive measures included low dose subcutaneous heparin in 55 patients (since 1978), and intermittent pneumatic compression boots in 29 (since 1985). Four patients developed life-threatening complications including myocardial infarction (two patients), congestive heart failure (one patient), and pulmonary embolism (one patient). Two of these four patients died; one with a myocardial infarction and the other with pulmonary embolism. The morbidity rate was thus 4.2% (4 out of 96), and the mortality was 2.1% (2 out of 96). Although the four serious complications occurred within 30 days of the procedure, only one complication and one death occurred during treatment. There was no correlation between occurrence of complications and medical risk factors, type and duration of anesthesia, or type and duration of implant. CONCLUSIONS: There is a low incidence of complications associated with conventional low dose rate brachytherapy. The procedure is well tolerated in patients with medically inoperable Stage I endometrial cancer. In comparison to the predicted serious complication rate of surgery in these patients, the number of life-threatening complications from brachytherapy appears to be quite acceptable.

Effects of volatile anesthetics on acetylcholine-induced relaxation in the rabbit mesenteric resistance artery.

BACKGROUND: Vascular endothelium plays an important role in the regulation of vascular tone. Volatile anesthetics have been shown to attenuate endothelium-mediated relaxation in conductance arteries, such as aorta. However, significant differences in volatile anesthetic pharmacology between these large vessels and the small vessels that regulate systemic vascular resistance and blood flow have been documented, yet little is known about volatile anesthetic action on endothelial function in resistance arteries. Furthermore, endothelium-dependent relaxation mediated by factors other than endothelium-derived relaxing factor (EDRF) has recently been recognized, and there is no information available regarding volatile anesthetic action on non-EDRF-mediated endothelium-dependent relaxation. METHODS: Employing isometric tension recording and microelectrode methods, the authors first characterized the endothelium-dependent relaxing and hyperpolarizing actions of acetylcholine (ACh) in rabbit small mesenteric arteries, and tested the sensitivities of these actions to EDRF pathway inhibitors and K+ channel blockers. They then examined the effects of the volatile anesthetics isoflurane, enflurane, and sevoflurane on ACh-induced endothelium-dependent relaxation that was sensitive to EDRF inhibitors and that which was resistant to the EDRF inhibitors but sensitive to blockers of ACh-induced hyperpolarization. The effects of the volatile anesthetics on endothelium-independent sodium nitroprusside (SNP)-induced relaxation were also studied. RESULTS: Acetylcholine concentration-dependently caused both endothelium-dependent relaxation and hyperpolarization of vascular smooth muscle. The relaxation elicited by low concentrations of ACh (< or = 0.1 microM) was almost completely abolished by the EDRF inhibitors NG-nitro-L-arginine (LNNA), oxyhemoglobin (HbO2), and methylene blue (MB). The relaxation elicited by higher concentrations of ACh (> or = 0.3 microM) was only attenuated by the EDRF inhibitors. The remaining relaxation, as well as the ACh-induced hyperpolarization that was also resistant to EDRF inhibitors, were both specifically blocked by tetraethylammonium (TEA > or = 10 mM). Sodium nitroprusside, a NO donor, produced dose-dependent relaxation, but not hyperpolarization, in the endothelium-denuded (E[-]) strips, and the relaxation was inhibited by MB and HbO2, but not TEA (> or = 10 mM). One MAC isoflurane, enflurane, and sevoflurane inhibited both ACh relaxation that was sensitive to the EDRF inhibitors and the ACh relaxation resistant to the EDRF inhibitors and sensitive to TEA, but not SNP relaxation (in the E[-] strips). An additional finding was that the anesthetics all significantly inhibited norepinephrine (NE) contractions in the presence and absence of the endothelium or after exposure to the EDRF inhibitors. CONCLUSIONS: The results confirm that ACh has a hyperpolarizing action in rabbit small mesenteric resistance arteries that is independent of EDRF inhibitors but blocked by the K+ channel blocker TEA. The ACh relaxation in these resistance arteries thus appears to consist of distinct EDRF-mediated and hyperpolarization-mediated components. Isoflurane, enflurane, and sevoflurane inhibited both components of the ACh-induced relaxation in these small arteries, indicating a more global depression of endothelial function or ACh signaling in endothelial cells, rather than a specific effect on the EDRF pathway. All these anesthetics exerted vasodilating action in the presence of NE, the primary neurotransmitter of the sympathetic nervous system, which plays a major role in maintaining vasomotor tone in vivo. This strongly indicates that the vasodilating action of these anesthetics probably dominates over their inhibitory action on the EDRF pathway and, presumably, contributes to their known hypotensive effects in vivo. Finally, the vasodilating action of these anesthetics is, at least in part, independent from endothelium.

Endothelium-independent vasoconstricting and vasodilating actions of halothane on rat mesenteric resistance blood vessels.

BACKGROUND: Whether volatile anesthetics produce changes in vascular resistance and blood flow because of direct effects on vascular tissue is unclear. Direct vasoconstricting and vasodilating actions have been demonstrated in isolated conductance arteries in vitro, but there is little information regarding direct effects on the small vessels that mediate resistance and flow changes in vivo. METHODS: We investigated the actions of halothane on 50-200 microM branches of the rat mesenteric artery that were cannulated and studied in vitro. The vessels were pressurized to 60 mmHg, and vascular dimensions were continuously monitored using a computer-based real-time image analysis system. The vessel bath was perfused with HCO3(-)-buffered saline (37 degrees C) equilibrated with 95% O2/5% CO2 (+/- halothane). The vascular endothelium was mechanically removed before cannulation in some vessels. RESULTS: In unstimulated vessels, halothane had a concentration-dependent vasoconstricting action (EC50 = 0.45 mM approximately 1.5 vol% at 37 degrees C) that was largely transient and was similar to that produced by caffeine. Both halothane and caffeine constrictions were unaffected by bath [Ca2+], nifedipine (1 microM) or Cd2+ (100 microM) and were abolished by ryanodine (10 microM). In addition, caffeine responses were attenuated by halothane in a concentration-dependent manner (EC50 = 1.6 mM). In vessels preconstricted with KCl (40 mM) or phenylephrine (10(-6) M), halothane produced transient constriction followed by concentration-dependent vasodilation. Ryanodine, which abolished halothane constrictions, had little effect on the amplitude of KCl- or phenylephrine-induced constrictions or the vasodilating action of halothane. Removal of the endothelium likewise had little effect on the vasoconstricting or the vasodilating actions of halothane in unstimulated, KCl- or phenylephrine-constricted vessels. Halothane completely relaxed KCl and phenylephrine constrictions with EC50 values of 0.36 mM (1.2% at 37 degrees C) and 0.75 mM (2.5%), respectively, in intact vessels before ryanodine; 0.25 mM (0.8%) and 0.59 mM (1.9%) in intact vessels after ryanodine; and 0.52 mM (1.7%) and 0.67 mM (2.2%) in endothelium-denuded vessels. CONCLUSIONS: Halothane has endothelium-independent vasoconstricting and vasodilating actions in isolated mesenteric resistance blood vessels. The vasoconstricting action appears to involve halothane-induced Ca2+ release from caffeine/ryanodine-sensitive intracellular store(s). The vasodilating action in phenylephrine- or KC1-constricted vessels is independent of the Ca(2+)-releasing action and most likely involves an effect(s) on sarcolemmal-dependent Ca2+ signaling (e.g., extracellular Ca2+ influx) and/or Ca2+ activation of contractile proteins. The magnitude of both the vasoconstricting and the vasodilating actions of halothane in these vessels at clinically relevant concentrations suggests these direct actions contribute to the overall cardiovascular effects of halothane in vivo.

"Caged" phenylephrine: development and application to probe the mechanism of alpha-receptor-mediated vasoconstriction.

A "caged" analogue of the alpha-adrenergic receptor agonist phenylephrine (PE) was prepared by exploiting the 2-nitrobenzyl protecting group and using a synthetic procedure developed to permit preferential derivatization at the amino group. On isolated adult rat mesenteric arterioles, caged-PE had no measurable effects at concentrations up to 100 microM; 0.5-ms light flashes in the presence of caged-PE, however, produced marked and dose-dependent vasoconstriction. Flash-induced vasoconstrictions were blocked by the alpha-receptor antagonist phentolamine and were unaffected by the beta-receptor antagonist propranolol, indicating that the light-induced responses reflect the selective activation of alpha-adrenergic receptors. After a single flash, a large transient decrease in vessel diameter was recorded, and in most vessels, this was followed by a smaller, sustained constriction. The sustained component of the contraction was selectively eliminated when Ca2+ was removed from the bath, which suggests that different mechanisms underlie the transient and the sustained responses to PE. The responses to single flashes of varying intensities occurred with a mean latency of 460 ms, which is consistent with the intermediacy of several steps between alpha-receptor activation and contraction. We anticipate that it will be possible to extend this approach to develop caged analogues of other neurotransmitters for mechanistic and kinetic studies.

Two functionally distinct 4-aminopyridine-sensitive outward K+ currents in rat atrial myocytes.

In the experiments here, the detailed kinetic properties of the Ca(2+)-independent, depolarization-activated outward currents (Iout) in enzymatically dispersed adult rat atrial myocytes were studied. Although there is only slight attenuation of peak Iout during brief (100 ms) voltage steps, substantial decay is evident during long (10 s) depolarizations. The analyses here reveal that current inactivation is best described by the sum of two exponential components, which we have termed IKf and IKs to denote the fast and slow components, respectively, of Iout decay. At all test potentials, IKf inactivates approximately 20-fold more rapidly than IKs. Neither the decay time constants nor the fraction of Iout remaining at the end of 10-s depolarizations varies over the potential range of 0 to +50 mV, indicating that the rates of inactivation and recovery from inactivation are voltage independent. IKf recovers from inactivation completely, independent of the recovery of IKs, and IKf recovers approximately 20 times faster than IKs. The pharmacological properties of IKf and IKs are similar: both components are sensitive to 4-aminopyridine (1-5 mM) and both are relatively resistant to externally applied tetraethylammonium (50 mM). Taken together, these findings suggest that IKf and IKs correspond to two functionally distinct K+ currents with similar voltage-dependent properties and pharmacologic sensitivities, but with markedly different rates of inactivation and recovery from inactivation. From the experimental data, several gating models were developed in which voltage-independent inactivation is coupled either to channel opening or to the activation of the individual channel subunits. Experimental testing of predictions of these models suggests that voltage-independent inactivation is coupled to activation, and that inactivation of only a single subunit is required to result in functional inactivation of the channels. This model closely approximates the properties of IKf and IKs, as well as the composite outward currents, measured in adult rat atrial myocytes.

Cloning and expression of a rat cardiac delayed rectifier potassium channel.

We have cloned a cDNA (designated RAK) coding for a delayed-rectifier K current (IRAK) from adult rat heart atrium and expressed it in Xenopus oocytes. RAK differs from the cloned rat brain K current, BK2 [McKinnon, D. (1989) J. Biol. Chem. 264, 8230-8236], by one amino acid at residue 411. RAK expressed in oocytes compares closely to the intrinsic adult rat atrial delayed-rectifier current measured by using whole-cell recording of single isolated cells. Northern blot analysis confirmed the presence of the channel in adult rat atrium, and to a lesser extent, in rat ventricle. IRAK activates with time constants ranging from 58 ms at -20 mV to 6 ms at +60 mV and does not show significant inactivation over 800 ms. It is blocked by 4-aminopyridine greater than barium much greater than tetraethylammonium chloride, which is similar to the relative potencies of these blockers on the native delayed rectifier current. We conclude that the main delayed rectifier K current in adult rat atria is virtually identical to a neuronal delayed rectifier, BK2.

A novel type of depolarization-activated K+ current in isolated adult rat atrial myocytes.

To determine the types of voltage-gated K+ channels controlling action potential repolarization in atrial cells, we have characterized the properties of depolarization-activated K+ channels in isolated adult rat atrial myocytes using the whole cell patch-clamp recording technique. On membrane depolarization, Ca2(+)-independent outward K+ currents in these cells begin to activate at approximately -40mV. At all test potentials, the currents activate rapidly after a delay, and there is little or no decay of the peak outward current amplitude during brief (100 ms) depolarizations. In addition, the currents show little steady-state inactivation at membrane potentials negative to -60 mV. The currents are blocked effectively by 1-5 mM 4-aminopyridine but are relatively insensitive to extracellular tetraethylammonium at concentrations up to 50 mM. Based on the measured time- and voltage-dependent properties and the pharmacological sensitivity of the currents, we suggest that the depolarization-activated K+ channels underlying the macroscopic currents in adult rat atrial myocytes are distinct from those described previously in other myocardial preparations, including adult rat ventricular myocytes. Interestingly, the outward K+ currents characterized here in isolated adult rat atrial myocytes are remarkably similar to those of several recently described "delayed rectifier" K+ channel genes isolated from rat brain cDNA libraries and expressed in Xenopus oocytes, suggesting that similar K+ currents are likely present in cells of the mammalian central nervous system.

Attenuation of vasopressin-mediated coronary constriction and myocardial depression in the hypoxic heart.

To investigate the ability of arginine vasopressin (AVP) to compete with metabolic vasodilatory factors in the coronary circulation, we examined the coronary vascular and myocardial effects of AVP in isolated working rat hearts during normoxic and hypoxic perfusion. In normoxic hearts, AVP treatment (777 +/- 67 pg/ml) reduced coronary flow by 38.4 +/- 2.6%. Myocardial function was also significantly decreased by AVP whereas efficiency significantly increased. In contrast, the same dose of AVP administered to hypoxic hearts resulted in substantially smaller effects on coronary flow (-11.5 +/- 2.8%), myocardial function, and efficiency. In hearts treated first with AVP and then with hypoxia, the greater degree of coronary vasodilation compared with that observed in hearts treated with hypoxia alone also indicated an antagonizing effect of hypoxia on AVP-mediated coronary constriction. It was also noted that the hypoxia treatment alone resulted in reductions of O2 supply and consumption identical to those produced by AVP treatment during normoxia. However, hypoxia was associated with a significantly greater effect on myocardial function and, in contrast to the effect of AVP, a marked reduction in efficiency. The rate of lactate release was greater during hypoxia alone (2.07 +/- 0.08 mumol/min) than with AVP treatment during normoxia (0.76 +/- 0.05 mumol/min). These results indicate that the effect of AVP on the coronary vessels, as well as its effect on the myocardium, is significantly attenuated during hypoxia. In addition, AVP-constricted vessels appear to retain considerable vasodilatory reserve despite evidence of ischemic conditions. Thus, although the effects of AVP resemble ischemia, the increased efficiency and the relatively small effect of AVP on contractile function, as well as the preserved vasodilatory reserve, suggest otherwise. A physiological explanation for these observations is proposed wherein the constricting effects of AVP modulate the effects of autoregulatory factors such that blood flow requirements are minimized while allowing preservation of adequate blood flow for vital tissue function.