Exercise training improves cardiac function after ischemia in the isolated, working rat heart.

The aim of this study was to determine whether exercise training produces a myocardium intrinsically more tolerant to ischemic-reperfusion injury. Male Fischer 344 rats were treadmill trained for 11-16 wk at one of the following intensities: LOW (20 m/min, 0% grade, 60 min/day), moderate (MOD; 30 m/min, 5% grade, 60 min/day) or intensive (INT; 10 bouts of alternating 2-min runs at 16 and 60 m/min, 5% grade). Cardiac function was evaluated both before and after 25 min of global, zero-flow ischemia in the isolated, working heart model. Compared to hearts from sedentary (SED) rats, postischemic cardiac output (CO) and work were significantly higher in all trained groups. Percent recovery of CO (relative to preischemia) was 36.0 +/- 7.1 in SED and 61.2 +/- 6.5, 68.1 +/- 9.3, and 73.2 +/- 5.0 in LOW, MOD, and INT, respectively. Postischemic increases in stroke volume with increased preload and cardiac work at high work load were significantly higher in INT compared with SED. Coronary flow during initial retrograde reperfusion was significantly enhanced with training and correlated with subsequent recovery of CO (R2 = 0.613). Furthermore, trained hearts had higher phosphocreatine (P less than 0.05) and ATP (P less than 0.01) contents after 45 min reperfusion. It is concluded that exercise training results in an intrinsic myocardial adaptation, allowing greater recovery of cardiac pump function after global ischemia in the isolated rat heart.


Leukocyte adhesion in local versus hemorrhage-induced ischemia.

The objective of this study was to compare the leukocyte-endothelial cell adhesive interactions elicited in postcapillary venules by either local ischemia-reperfusion or hemorrhage-reperfusion. Leukocyte rolling, adherence, and emigration were monitored in cat mesenteric venules exposed to an 85% reduction in blood flow (induced by either hemorrhage or local restriction of arterial inflow) for 1 h, followed by 1 h reperfusion. Leukocyte-endothelial cell interactions, venular diameter, and red blood cell velocity were measured during baseline, ischemia, and reperfusion periods. Both local and hemorrhage-induced ischemia reperfusion caused a reduction in leukocyte rolling velocity and increases in leukocyte adherence and emigration. Quantitatively, the adherence and emigration responses in both ischemia models were nearly identical. However, the two models differed in their response to immunoneutralization of the leukocyte adhesion glycoprotein CD11/CD18 with monoclonal antibody (MAb) IB4. The MAb had a more profound effect in attenuating leukocyte adherence and emigration in the local ischemia model. These results indicate that different factors may contribute to leukocyte-endothelial cell adhesive interactions observed in local vs. systemic models of ischemia-reperfusion.


Effect of abrupt changes in ventricular loading on repolarization induced by transient aortic occlusion in humans.

We have investigated the influence of ventricular loading on repolarization from beat to beat in the human heart. Sixteen patients undergoing routine coronary artery surgery were studied. Left ventricular epicardial monophasic action potentials and local electrograms were recorded during acute changes in ventricular loading induced by transient aortic occlusion. Monophasic action potential duration shortened (P less than 0.0001) and returned to control values within one or two beats after release (P less than 0.0001). Values at 90% repolarization were 325 +/- 31 ms preocclusion, 311 +/- 29 ms during occlusion, 326 +/- 32 ms postocclusion. The Q-T interval of the local epicardial electrogram shortened during occlusion (P less than 0.001) and returned to control values after release (P greater than 0.0001): 396 +/- 44 ms preocclusion, 379 +/- 41 ms during occlusion, and, 399 +/- 42 ms postocclusion. A significant correlation was obtained between changes in peak systolic pressure and changes in monophasic action potential duration (R = 0.96; P less than 0.0001 at 90% repolarization). A significant correlation was also observed between changes in peak systolic pressure and the Q-T interval of the local electrogram (R = 0.91; P less than 0.0001). This study shows that abrupt changes in ventricular loading from one beat to the next induce significant changes in the timing of ventricular repolarization. These results may well be relevant to the initiation of arrhythmia by a single ventricular ectopic beat, particularly under pathological conditions.


Thoracic aortic pressure-flow relationships and vascular impedance in fetal sheep.

Upper descending thoracic aortic pressures recorded from near-term fetal sheep resembled those recorded in mature animals, but diastolic thoracic aortic flows were much greater than those reported for adults. Furthermore, the pressure wave was not amplified during transmission along the aorta, as it is in adults. Descending thoracic impedance fell with increasing frequency to approximately 25% of resistance to steady flow and then showed oscillations typical of systems in which pulse wave reflections are important. Angiotensin II and norepinephrine infusions enhanced impedance oscillations and caused changes in apparent phase velocity in the aorta that were consistent with increased wave reflections. Angiotensin also increased aortic characteristic impedance from 0.0152 +/- 0.0013 to 0.0230 +/- 0.0024 mmHg.min.ml-1. The effects of nitroprusside infusions indicated reduced wave reflection effects. Embolization of the placenta and hindlimbs with 50-microns microspheres was used to elevate vascular resistance in these beds. Embolization caused aortic flow to fall to zero by late diastole, and changes in impedance and apparent phase velocity suggested increased wave reflections from the periphery. Characteristic impedance was increased to 0.0209 +/- 0.0031 mmHg.min.ml-1.


Chronic captopril and losartan (DuP 753) administration in rats with high-output heart failure.

We investigated the role of atrial natriuretic factor (ANF) and the renin-angiotensin system as well as the effects of losartan in rats with aortocaval (AC) shunts. Right atrial and left ventricular end-diastolic pressures (LVEDP) were higher and mean arterial blood pressure (MAP) was lower in AC shunt animals than in their controls. AC shunt rats presented marked cardiac hypertrophy, decreased right atrial ANF concentration, and increased ventricular ANF content and concentration. Plasma ANF levels were elevated, and hematocrit was lower in AC shunt animals than in controls. Captopril or losartan treatment decreased MAP and returned LVEDP to sham-operated control values. A clear regression of cardiac hypertrophy was evident in both treated AC shunt groups, with plasma ANF levels tending to follow those in sham-operated rats. Plasma COOH-terminal ANF levels were decreased and urinary volume and hematocrit were increased in losartan-treated AC shunt animals. We conclude that chronic angiotensin converting enzyme inhibition and angiotension II receptor antagonism improved hemodynamic conditions, diminished water retention, reversed cardiac hypertrophy, and restored plasma and tissue ANF to more "normal" levels in rats with moderate high-output heart failure.


Mechanical performance of spared myocytes after myocardial infarction in rats: effects of captopril treatment.

To determine the effects of myocardial infarction-induced left ventricular failure on the mechanical characteristics of the remaining viable myocytes, coronary arterial occlusion was performed in rats, and cell function was examined 1 wk later. Moreover, to establish the mechanisms by which treatment with angiotensin-converting enzyme inhibitors ameliorates cardiac dynamics, captopril was administered immediately after surgery, and the contractile behavior of the unaffected cells was similarly analyzed 7 days later. The severe impairment in left-side pump function was found to be associated with a decrease in the velocities of myocyte shortening and relengthening and peak shortening despite a prolongation of contraction duration. In addition, a uniform property was recognized in myocytes from infarcted and noninfarcted hearts. Longer cells manifested greater velocity of shortening, whereas wider cells of identical length exhibited depressed shortening velocity. After infarction, the depression in cell contractility coupled with lateral expansion of myocytes exceeded the influence on cell mechanical behavior linked to myocyte lengthening leading to an overall decrease in contractility of the hypertrophied cells. Captopril therapy preserved, in part, the ability of myocytes to shorten and relengthen, which was accompanied by a decrease in the lateral and longitudinal expansions of these cells.


Cardiac venous endothelial dysfunction after myocardial ischemia and reperfusion in dogs.

Endothelial dysfunction is a prominent occurrence in coronary arteries after myocardial ischemia and reperfusion. However, this has not been studied in coronary veins. Endothelium-dependent vasorelaxation was studied in cardiac venous rings obtained from dogs subjected to 60 min of coronary artery occlusion followed by 270 min of reperfusion, as well as from dogs subjected to sham ischemia-reperfusion. Myocardial ischemia resulted in a 96 +/- 2% decrease in coronary flow to the ischemic area 60 min after occlusion of the left anterior descending (LAD) coronary artery, which led to a significant degree of cardiac necrosis (i.e., 32.9 +/- 3.9% of area at risk). Cardiac venous rings isolated from sham ischemia-reperfusion dogs relaxed 68 +/- 3% to 200 microM ADP, 65 +/- 3% to 2 microM A23187, and 76 +/- 4% to 200 microM sodium nitrite (NaNO2). Corresponding values for cardiac venous rings isolated from ischemic-reperfused dogs were 32 +/- 3% for 200 microM ADP (P less than 0.01 vs. sham), 31 +/- 3% for 2 microM A23187 (P less than 0.01 vs. sham), and 74 +/- 3% for 200 microM NaNO2 (NS from sham). In rings from control dogs, vasorelaxation to ADP and A23187 was markedly inhibited by 4 mM NG-methyl-L-arginine (L-NMMA) and 10 methylene blue and restored after NG-nitro-L-arginine by 3 mM L-arginine. These results demonstrate that a significant degree of endothelial dysfunction occurred in cardiac venous rings after ischemia and reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)


Na+ efflux mechanisms in ventricular myocytes: measurement of [Na+]i with Na(+)-binding benzofuran isophthalate.

We characterized the Na(+)-sensitive dye benzofuran isophthalate (SBFI) with fluorescence microscopy in isolated, adult rat ventricular myocytes. When cells were loaded with SBFI by incubation with 10 microM of the acetoxymethyl ester, fluorescence excitation spectra were markedly attenuated below 340 nm and the isoemissive point was blue shifted by approximately 25 nm when compared with spectra from SBFI acid in buffered solutions. Fluorescence intensity (49 +/- 3%) was partially released by permeabilization of the sarcolemma with digitonin, suggesting that one-half of the dye molecules are sequestered subcellularly in a compartment shown most likely to be mitochondrial. Intracellular Na+ concentration ([Na+]i) was determined by in situ calibration using cation-selective ionophores and was found to be 14 +/- 2 mM in cells studied at 37 degrees C. The relative importance of Na+ efflux mechanisms in myocytes was investigated. Substitution of Ca2+ and Mg2+ with EGTA in the superfusing medium resulted in a reversible rise of [Na+]i from 13 +/- 2 to 31 +/- 5 mM, which was blocked by 1 microM verapamil. Cellular efflux of Na+ after loading in this manner was found to be insensitive to blockade of Na(+)-Ca2+ exchange but was abolished when Na(+)-K(+)-ATPase was inhibited with zero extracellular K+ concentration. We conclude that SBFI can be used to measure [Na+]i in ventricular cells nondestructively and without impalement. Na+ efflux after loading by Ca2+ and Mg2+ withdrawal is mediated by the Na+ pump with no measurable contribution from Na(+)-Ca2+ exchange.


Hemodynamic effects of exogenous nitric oxide in ovine transitional pulmonary circulation.

To determine the hemodynamic effects of exogenous nitric oxide (NO) on the immature pulmonary circulation, we studied the response to NO inhalation in 19 mechanically ventilated late-gestation ovine fetuses in three separate protocols. In protocol 1, we examined the relative effects of 1) mechanical ventilation while maintaining fetal arterial O2 tension (PaO2) constant [fractional inspired O2 concentration (FIO2) less than 0.10)], 2) NO inhalation [5-20 parts per million (ppm)] at fetal PaO2, and 3) high FIO2 (1.00) (n = 7). NO increased left pulmonary artery blood flow (Qlpa) in a dose-dependent fashion, from 254 +/- 62 (baseline) to 398 +/- 49 ml/min with 20 ppm NO (P less than 0.001). The response of Qlpa to a FIO2 equal to 1.00 was not different from NO alone. Systemic arterial pressure was not affected by NO. In protocol 2 we studied the effects of prolonged NO inhalation (2 h, 20 ppm) during mechanical ventilation with low FIO2 (n = 4). NO increased Qlpa from 267 +/- 92 to 468 +/- 75 ml/min at 10 min of NO inhalation (P less than 0.001). The increase in Qlpa was sustained during the entire 2-h study period. In protocol 3 we measured left ventricular output (LVO), systemic vascular resistance (SVR), and ductus arteriosus shunting using radiolabeled microspheres (n = 8) during baseline mechanical ventilation and 20 ppm NO inhalation. LVO and SVR were not significantly different in the two study periods; however, the percentage of LVO that reached the lungs (predominantly left-to-right shunting across the ductus arteriosus) increased from 18 +/- 5 to 43 +/- 4% during NO inhalation.(ABSTRACT TRUNCATED AT 250 WORDS)


Pulmonary and systemic vascular smooth muscle mechanical characteristics in newborn sheep.

To investigate the hypothesis that the higher pulmonary vascular resistance in newborn sheep is the result of developmental differences in the vascular muscle mechanical properties, we evaluated pulmonary arteries from newborn and adult sheep and compared them with their respective systemic counterparts (common carotid arteries). The newborn pulmonary artery mechanical stress (13.0 +/- 1.4 mN/mm2) and shortening capacity (11.4 +/- 1.1% of optimal length) were lower (P less than 0.01) than in the adult (20.4 +/- 2.5 and 15.6 +/- 1.3, respectively). The adult carotid artery muscle developed a greater stress (97.6 +/- 18.5 mN/mm2) than the newborn (40.7 +/- 5.0; P less than 0.01), whereas no age differences in shortening capacity were observed (newborn = 19.4 +/- 1.7; adult = 18.4 +/- 1.5% of optimal length). The contraction half-time was similar for the pulmonary and carotid arteries and was not affected by age, whereas the relaxation half-times of the newborn pulmonary (30.7 +/- 2.9 s) and carotid artery (23.3 +/- 1.5) were greater than in the adult (24.9 +/- 2.9 and 14.6 +/- 1.4, respectively; P less than 0.01). The myosin contents of the pulmonary and carotid arteries, as an indicator of the tissue muscle mass, were similar and did not change with age. In conclusion, while the lower stress and shortening capacity of the newborn pulmonary arteries limit their maximum capacity to vasoconstrict, the significantly greater relaxation time of their vascular muscle, a new observation, may account for the higher resistance to blood flow after birth.


Ischemic preconditioning attenuates acidosis and postischemic dysfunction in isolated rat heart.

The hypothesis that brief ischemia (preconditioning) protects the isolated heart from prolonged global ischemia was tested. Isovolumic rat hearts were preconditioned with either 5 min of ischemia followed by 5 min of perfusion (P1) or two 5-min episodes of ischemia separated by 5 min of perfusion (P2). Control hearts received no preconditioning. All hearts received 40 min of sustained ischemia and 30 min of reperfusion. Preconditioning (P1 or P2) significantly (P less than 0.0005) improved recovery of the rate-pressure product; percentage recoveries were 17.8 +/- 3.2 (n = 14), 59.9 +/- 5.5 (n = 6), and 46.4 +/- 4.7 (n = 8) for control, P1, and P2, respectively. Improved functional recovery of preconditioned hearts was associated with reduced end-diastolic pressure and improved myocardial perfusion. During the 40-min ischemic period, myocardial pH decreased from approximately 7.4 to 6.3 +/- 0.1 (n = 7) in the control hearts and to 6.7 +/- 0.1 (n = 7) in the preconditioned hearts (P less than 0.01). Also during the 40-min ischemic period, myocardial lactate (expressed as nmol/mg protein) increased to 146 +/- 11 (n = 7) and 101 +/- 12 (n = 8) in control and preconditioned hearts, respectively (P less than 0.02). The results demonstrate that a brief episode of ischemia can protect the isolated rat heart from a prolonged period of ischemia. This protection is associated with decreased tissue acidosis and anaerobic glycolysis during the sustained ischemic period.


Comparison of protein lymph flux and extravascular uptake in skin during increased venous pressure.

The hypothesis that the albumin reflection coefficient is underestimated from measurements of lymph protein flux because of a prolonged interstitial washout was tested in the hindleg of anesthetized rabbits. In heel skin, the initial extravascular uptake for labeled albumin and immunoglobulin G (IgG) was compared with prenodal lymph flux for native albumin and IgG. Venous pressure was increased in one leg for 4 h, while the contralateral leg was the control. The extravascular uptake for labeled albumin was 67% greater than control during the fourth hour of increased venous pressure. Lymph albumin flux was 2.3 times control, indicating a small but significant washout of interstitial albumin. The magnitude of the interstitial washout for IgG was less than that for albumin. Using the relationship between the change in extravascular uptake and the change in lymph flow, the reflection coefficients for albumin and IgG were both 0.91. The reflection coefficient for albumin using lymph protein flux was lower because of a continued interstitial washout.


31P-NMR of high-energy phosphates in perfused rat heart during metabolic acidosis.

Intracellular pH (pHi), intracellular free magnesium concentration ([Mg2+]i), and high-energy phosphates in Langendorff perfused rat hearts were evaluated by 31P-nuclear magnetic resonance (NMR) during metabolic acidosis. During acidosis, cardiac pHi approached that of the perfusing solution (pH approximately 6.7) and [Mg2+]i increased. In hearts perfused with glucose as the sole carbon source, the ratio of [phosphocreatine] to [ATP] decreased during acidosis. In contrast, in hearts supplemented with pyruvate (either 2.8 or 10 mM) this ratio increased during acidosis. Oxygen consumption decreased in hearts perfused with glucose only and with pyruvate-glucose. Using the creatine kinase equilibrium constant, we find that [MgADP] is significantly decreased in pyruvate-perfused hearts but is not significantly altered in glucose-perfused hearts during metabolic acidosis. These data indicate that [MgADP] may be the regulator of cardiac oxidative phosphorylation in the presence of excess pyruvate; however, during metabolic acidosis in hearts perfused with glucose only, ATP synthesis appears limited by the availability of pyruvate via glycolysis.


Fetal cardiac bypass alters regional blood flows, arterial blood gases, and hemodynamics in sheep.

Successful fetal cardiac bypass might allow prenatal correction of some congenital heart defects. However, previous studies have shown that fetal cardiac bypass may result in impaired fetal gas exchange after bypass. To investigate the etiology of this impairment, we determined whether fetal cardiac bypass causes a redistribution of fetal regional blood flows and, if so, whether a vasodilator (sodium nitroprusside) can prevent this redistribution. We also determined the effects of fetal cardiac bypass with and without nitroprusside on fetal arterial blood gases and hemodynamics. Eighteen fetal sheep were studied in utero under general anesthesia. Seven fetuses underwent bypass without nitroprusside, six underwent bypass with nitroprusside, and five were no-bypass controls. Blood flows were determined using radionuclide-labeled microspheres. After bypass without nitroprusside, placental blood flow decreased by 25-60%, whereas cardiac output increased by 15-25%. Flow to all other fetal organs increased or remained unchanged. Decreased placental blood flow after bypass was accompanied by a fall in PO2 and a rise in PCO2. Nitroprusside improved placental blood flow, cardiac output, and arterial blood gases after bypass. Thus fetal cardiac bypass causes a redistribution of regional blood flow away from the placenta and toward the other fetal organs. Nitroprusside partially prevents this redistribution. Methods of improving placental blood flow in the postbypass period may prove critical to the success of fetal cardiac bypass.


Low-dose endothelin-1 potentiates volume-induced secretion of atrial natriuretic factor.

To evaluate the role of calcium in volume-induced secretion of atrial natriuretic factor (ANF), effects of verapamil and endothelin-1 (ET-1), both known to influence free intracellular calcium, were studied during saline infusion in seven conscious instrumented dogs. Fifteen minutes after intravenous injection of placebo or verapamil (0.25 mg/kg) or during continuous ET-1 infusion (at 5 ng.kg-1.min-1), saline (10% body wt) was infused during 40 min. Saline produced a rise (P less than 0.001) of plasma ANF from 28.1 +/- 6.3 to 50.4 +/- 12.9 pM after placebo, from 30.2 +/- 6.1 to 51.1 +/- 8.5 pM after verapamil, and from 31.2 +/- 6.1 to 81.0 +/- 12.9 pM with ET-1. This increase was comparable after placebo and verapamil, but was 80% greater with ET-1 (P less than 0.02). Plasma ET-1, unchanged after placebo, rose (P less than 0.001) from 1.7 +/- 0.5 to 38.3 +/- 9.2 pM with ET-1. In the three experiments, heart rate and left atrial pressure (LAP) increased (P less than 0.001) similarly. The linear relation between ANF and LAP was steeper with ET-1 than with saline or verapamil (both P less than 0.05), indicating that the enhanced ANF secretion with ET-1 was occurring at all levels of LAP. Thus volume-induced secretion of ANF is not suppressed by verapamil, but is directly enhanced by low-dose ET-1, known to activate the phosphoinositide pathway.


Concurrent increases in regional hematocrit and blood flow in diabetic rats: prevention by sorbinil.

These studies were undertaken to investigate the relationship between regional hemodynamic and hemorheological changes in the microvasculature of diabetic rats. Diabetes was induced in male Sprague-Dawley rats by injection of streptozotocin (55 mg/kg body wt). Control rats were injected with vehicle (sodium citrate buffer). A subgroup of diabetic rats was treated with an aldose reductase inhibitor (sorbinil) added to the diet in an amount to provide a daily dose of approximately 0.2 mmol.kg-1.day-1. Three weeks later all animals were anesthetized with thiobutabarbital sodium (Inactin, 100 mg/kg injected intraperitoneally) for assessment of blood flow (by injection of 15 microns microspheres) and regional hematocrit (determined by isotope-dilution techniques using 51Cr-labeled red blood cells and 125I-labeled bovine serum albumin) in selected tissues. The hematocrit in arterial blood samples was identical (approximately 46%) in controls and in diabetics. Regional hematocrits were much lower than arterial hematocrits in control rats and ranged from approximately 20% in ocular tissues, sciatic nerve, diaphragm, and skin to approximately 30% in brain, skeletal muscle, heart, and fat. Hematocrits of diabetic rats were markedly increased in ocular tissues, sciatic nerve, and skin but not in brain, heart, or skeletal muscle. These increases in regional hematocrit were associated with increases in blood flow and were largely prevented by sorbinil. Diabetes induced significant decreases in the mean transit times for whole blood and erythrocytes in all tissues examined except brain, retina, and skin.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelin in thoracic inferior vena caval constriction model of heart failure.

Recent studies have found endothelin-1 (ET), a potent vasoconstrictor of endothelial cell origin, is increased in congestive heart failure (CHF) in plasma and correlates with increased atrial pressures. Additionally, it has been shown that, despite increased circulating ET concentrations in CHF, a decreased responsiveness to exogenous ET infusion exists in an experimental model of CHF induced by rapid pacing. The present study was designed to determine whether plasma ET would be increased in a model of low cardiac output congestive failure without elevated atrial pressures produced by thoracic inferior vena caval constriction (TIVCC). In addition, we sought to determine whether this model of low cardiac output would show a full vasoconstrictive response to exogenous ET infusion and whether atrial natriuretic factor (ANF) infused acutely to achieve pathophysiological concentrations would modulate the systemic and renal hemodynamic responses to exogenous ET. Dogs were studied in the anesthetized state at 8-9 days after the TIVCC or sham operation. Baseline ET levels were significantly increased in the TIVCC dogs (n = 14) compared with sham operated dogs (n = 6; 41.4 +/- 7.1 vs. 17.9 +/- 1.5 pg/ml, respectively; P less than 0.05). With exogenous ET infusion (5 ng.kg-1.min-1), an intact systemic and renal vasoconstrictor response in the TIVCC dogs was observed. ANF prevented the systemic vascular and arterial pressure responses to ET. However, the potent renal vasoconstricting action of ET was not prevented by ANF. These studies suggest that ET may play an important role in maintaining blood pressure in the setting of low cardiac output.(ABSTRACT TRUNCATED AT 250 WORDS)


Hypoxia induces endothelial cells to increase their adherence for neutrophils: role of PAF.

We investigated the interactions of polymorphonuclear neutrophils (PMN) and endothelial cells in myocardial ischemia using a hypoxia model. We exposed porcine aortic (PAEC) and porcine coronary microvessel (PCMEC) endothelial cells to 2% O2 for 2 h (PO2 = 53 mmHg) and measured the adherence of unstimulated neutrophils (PMN) to both control and hypoxia-conditioned endothelial cell monolayers. Hypoxia conditioning increased PMN adherence to PAEC and PCMEC by 51 and 101%, respectively, above control levels. The increase in PMN adhesion to PAEC was associated with a threefold increase in endothelial cell-associated platelet-activating factor (PAF) compared with control PAEC. The conditioned media from PAEC exposed to hypoxia also contained sixfold more PAF than control conditioned media, and it activated PMN to become adherent to untreated PAEC. The hypoxia-induced PAEC adhesion response was inhibited by preincubating PMN with the specific PAF receptor antagonist, L-659,989. We conclude that PAF is produced by cultured endothelial cells in response to hypoxia and that PAF generation is chiefly responsible for the increased adherence properties of hypoxia-conditioned endothelial cells. This response may play a major role in regulating PMN margination during myocardial ischemia.


Dynamic intramyocardial blood volume: evaluation with a radiological opaque marker method.

In this study the change of intramyocardial blood volume within one cardiac cycle was deduced from the time sequence of the volume delineated by radiopaque markers attached to the left ventricular wall. These data were compared with the change in the volume of the same region of myocardium estimated from the change in angiographic opacification of the heart wall. Five dogs were anesthetized, and four pairs of 2-mm-diameter lead beads were attached to the epicardial and endocardial regions. Two scans using the dynamic spatial reconstructor (a high-speed computed tomographic scanner) were performed during aortograms, one under control conditions and the other during adenosine infusion. The results have shown that the increases of myocardial volume defined by the beads (delta Vbead, % of volume at control) were comparable to the increase of intramyocardial blood volumes estimated by the increase in wall opacity (delta Vblood, % of muscle); delta Vblood = 0.93 delta Vbead + 1.54%; r = 0.987.


Measurement of biventricular septal-to-free wall diameters using sonomicrocrystals.

An ultrasonic system for the measurement of biventricular septal-to-free wall internal diameters in the dog was described. The ventricular endocardial ultrasonic microcrystals were implanted via the left and right atrial appendages and arranged in a straight line, with minimal invasion of the myocardium and pericardium. Reliable signals were obtained during right ventricular pressure overload and septal myocardial ischemia.


Suppression of motion artifacts in fluorescence spectroscopy of perfused hearts.

Fluorescence spectroscopy of beating hearts has been used previously to measure intracellular regulators of function. Unfortunately, heart motion could introduce a spurious motion artifact (MA), influencing the measured fluorescence intensity ("signal"). To suppress MA, a ratio (or difference) has been calculated previously between the signal and an intensity reference detected at a different wavelength ("reference"). However, no studies have attempted to evaluate or optimize the efficiency of MA suppression. MA suppression was evaluated using reflected excitation light or fluorescence as reference. In addition, the MA contribution to the intensity ratio from a fluorescent dye, indo-1, was quantified. A reflected light reference resulted in poor suppression of MA. The use of a fluorescence reference resulted in suppression that was inversely related to the detection wavelength difference (delta) between the reference and signal. Therefore optimal MA suppression was obtained using a fluorescence reference at a wavelength close to the signal. For delta = 60 nm, MA was suppressed from approximately 10 to less than 2%. Finally, suppressed MA (delta = 60 nm) accounted for less than 10% of the indo-1 ratio fluctuations.


Vertebrate intestine apical membrane mechanisms of organic nutrient transport.

This paper presents the current understanding of comparative vertebrate intestine basic mechanisms of brush-border membrane transport. Animals control the uptake of monosaccharides and amino acids at three levels: 1) mucosal hyperplasia increases uptake nonselectively, 2) individual enterocytes increase the transport capacity of specific transporter systems, and 3) the transporters themselves are modulated by solute and ion electrochemical gradients. In light of the current literature, This paper summarizes the kinetics, thermodynamics, and the physical arrangement of one mode of transport, the prototype Na(+)-solute cotransporter. The model presented is experimentally consistent with "preferred random" kinetics, with Na+ binding preferentially before solute at the extracellular face. In the case of glucose, the cotransporter system may be physically arranged in the membrane as a tetramer comprising 73,000 Da subunits. All vertebrates may have evolved with a similar mechanism, with particular variations reflecting selected arrangements from a pool of polypeptide sequence blocks. The same fundamental transport mechanisms may be observed in the intestines of animals ranging from lower vertebrates through humans.


Comparative aspects of lipid digestion and absorption: physiological correlates of wax ester digestion.

Our understanding of lipid digestion and absorption is largely derived from studies with glyceride-based dietary lipids. Yet for a great majority of marine species, wax esters (long-chain fatty alcohols esterified to long-chain fatty acids) are the dominant dietary neutral lipid. Many birds, especially seabirds (9 species) and some passerines (2 species), have a unique capacity for assimilating wax esters with higher efficiencies (greater than 90%) than that attainable by mammals (less than 50%). This unique capacity is correlated with several factors. One factor is an elevated intestinal bile salt concentration approaching 50 mM and a gallbladder concentration exceeding 600 mM. A second factor involves regular retrograde movement of duodenal contents to the gizzard. Thus not only is gastric emptying closely tied to the receptiveness of the duodenum for further handling of digesta as in mammals, but in birds the reflux returns the digesta (both gastric and duodenal) for further processing to the gizzard. A third key factor in wax ester utilization is a nearly equivalent hydrolysis of wax esters and triglycerides. Although similarities in fat digestion exist between birds and mammals, there do exist differences that make nonpolar lipid assimilation (i.e., wax esters) more efficient in birds, especially for seabirds.


Invertebrate gut diverticula are nutrient absorptive organs.

Physiological mechanisms of gastrointestinal absorption and secretion of organic and inorganic solutes among invertebrates remain severely underinvestigated. In this review we briefly discuss some of the physical and biological forces affecting solute transfer across epithelial cells and membranes of vertebrate and invertebrate guts. Next, we present some features of the described processes for sugar and amino acid transport in the tubular portion of gastrointestinal tracts of three major invertebrate groups: echinoderms, molluscs, and arthropods. A detailed discussion follows of recent nutrient transport studies using purified epithelial brush-border membrane vesicles of two invertebrate gut diverticula, the crustacean hepatopancreas and the starfish pyloric cecum. Lastly, transepithelial nutrient and ion transport studies of crustacean hepatopancreatic epithelial cell monolayers, grown in primary culture and mounted in flux chambers, are used to demonstrate the nature of absorption and secretion of solutes by this organ and to show the general applicability of these methods to structurally complex invertebrate organs.


Role of intestinal basolateral membrane in absorption of nutrients.

Organic solutes leave the intestinal epithelium and enter the circulation via specific facilitated carriers located in the basolateral membrane. In the case of glucose it is a low-affinity, high-capacity transport system that can adapt to the carbohydrate content of the diet. Chronic diabetes also promotes the exit of glucose, and in both cases the effect results from an increased density of carriers in the basolateral membrane. In contrast, a rapid upregulation of this system that can be induced within 30 min by hyperglycemia does not involve large changes in the amount of transporter protein. Similarly, the absorptive capacity of the small intestine from some amino acids can be influenced by events occurring at the basolateral membrane. In the case of dibasic amino acid absorption, exit from the epithelium is the rate-limiting step. The activity of the basolateral carrier can be increased almost 10-fold within 60 s by the addition of micromolar concentrations of the neutral amino acid leucine to either the lumen or the plasma. This response does not involve the second messenger adenosine 3',5'-cyclic monophosphate and may represent an allosteric modulation of the carrier. These observations are discussed in relation to the role of the basolateral membrane as a locus for controlling intestinal absorption of organic nutrients.


Molecular biology approaches to comparative study of Na(+)-glucose cotransport.

The rabbit intestinal Na(+)-glucose cotransporter has been cloned and sequenced. The cDNA encoding the cotransporter has been used in two general lines of research related to comparative aspects of Na(+)-glucose cotransport that are reviewed here. First, defined regions of the predicted amino acid sequence were used to raise antibodies, and the species distribution of epitopes recognized by those antibodies was investigated. Intestinal brush-border membranes from mammals, birds, an amphibian, and a reptile were all found to contain protein that were recognized by the antibodies in Western analysis. The cDNA encoding the rabbit intestinal Na(+)-glucose cotransporter was also used directly to examine the species distribution of related mRNA in Northern studies and to isolate new cDNAs encoding other Na(+)-glucose cotransporters. Northern blots revealed the presence of related mRNAs in intestines of mammals and a fish, as well as rabbit kidney and gills of marine mussels. The cDNAs encoding mammalian Na(+)-glucose cotransporters and bacterial Na(+)-dependent cotransporters for proline and pantothenate share sequence homology. There has been evolutionary conservation of the structure and function of the Na(+)-glucose cotransporter, and there appears to be a gene family that codes for the Na(+)-coupled cotransporters.


Tests of the adaptive modulation hypothesis for dietary control of intestinal nutrient transport.

According to the adaptive modulation hypothesis, an intestinal transporter should be repressed when its biosynthetic and other costs (of maintenance) exceed the benefits it provides. This leads to two contrasting predictions: transport of a sugar or amino acid worth calories should tend to be increased by its substrate, and transport of a vitamin should be modulated downwards by its substrate and upmodulated in deficiency. In a test of the first prediction, omnivorous desert iguanas eating alfalfa pellets (a high-carbohydrate diet) were compared with desert iguanas eating mealworms (a low-carbohydrate, higher-protein diet). In accord with the prediction, intact intestinal sleeves from the former group had higher rates of carrier-mediated D-glucose uptake/centimeter across the brush border than sleeves from the latter group. But in contrast to the first prediction, mealworm eaters had lower (not higher) proline uptake rates, and the ratio of glucose/proline uptake in the two groups did not differ. I review similar tests in 12 other species and show that overall the hypothesis has been quite robust with regard to the first prediction. Cases in which the hypothesis is rejected may reflect complications associated with changes in other dietary factors or phylogenetic constraints. In a test of the second prediction, uptake of the water-soluble vitamin choline was not increased in choline-deficient chicks, nor was it decreased in adults that have no dietary requirement for choline. I review similar tests for four other vitamins and five essential minerals. Dietary control of transport of the minerals and two of the vitamins seems to be in accord with the hypothesis. But transport rate for three vitamins (choline, pantothenic acid, ascorbic acid) seems not to be increased in deficiency. The best explanation seems to be that vitamin transport is modulated only if it is primarily by a carrier-mediated pathway.


Intestinal nutrient transport during ontogeny of vertebrates.

Patterns of transporter development are known for 12 species of vertebrates (2 fish, 1 amphibian, 1 bird, and 8 mammals), ranging from early gestation to adulthood. Although transporters for some nutrients (amino acids and some sugars) appear before the onset of external feeding, species differ in when and which transporters appear. Postnatal changes in the activities of different transporters are twofold: corresponding with shifts in the composition of a species' evolutionary diet, and the need to absorb ever-increasing quantities of food for growth and metabolism. The mechanisms responsible for the age-related shifts in rates of transport include changes in the densities, distribution, and types of transporters for specific nutrients and changes in the physiochemical characteristics of the intestinal brush-border membrane. The signals that trigger the age-related changes originate from internal preprogrammed sources and external sources, with both acting in concert to mediate intestinal development. Although much more is known for the brush-border hydrolases, recent studies with the pig suggest the ontogenetic development of hydrolases and transporters are regulated independently during early development.


Role of endothelium-derived nitric oxide in the renal hemodynamic response to amino acid infusion.

The present study was performed in anesthetized rats to compare the renal hemodynamic responses to mixed amino acids (M-AA) with those to L-arginine (L-Arg) and to examine the effect of endothelium-derived nitric oxide (EDNO) synthesis blockade on the M-AA-induced rise in renal plasma flow (RPF) and glomerular filtration rate (GFR). Intravenous infusion of both M-AA (Ser, Gly, Ala, and Pro, 0.71 mmol.100 g-1.min-1) and L-Arg (0.71 mmol.100 g-1.min-1) increased RPF and GFR. Peak increases in RPF for M-AA and L-Arg were 39.7% (P less than 0.05) and 63.4% (P less than 0.01), whereas GFR increases were 33.6% (P less than 0.05) and 46.7% (P less than 0.01, respectively). Outer cortical blood flow (OCBF) was increased with both treatments. Sodium excretion and urine flow were increased more with L-Arg than M-AA (both P less than 0.01). Infusion of the nitric oxide synthesis inhibitor, nitro-L-arginine (N-L-Arg, 20 micrograms.100 g-1.min-1) increased mean arterial pressure but decreased RPF 48.4% (P less than 0.001), OCBF 39.5% (P less than 0.001), GFR 37.8% (P less than 0.01), urine flow 29.8% (P less than 0.01), and sodium excretion 40.9% (P less than 0.01). When M-AA was administered after N-L-Arg, significant increases in OCBF, RPF, and GFR were observed (P less than 0.01); there was also an enhancement of sodium excretion and urine flow (both P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Maintenance of intestinal nutrient transport during hibernation.

We studied nutrient absorption across the brush-border membrane in jejunal tissues from active 13-lined ground squirrels and in hibernating squirrels that had not eaten for at least 6 wk. Body weights and jejunal wet weights per centimeter were significantly reduced in the hibernators. Rates of total and carrier-mediated uptake of 3-O-methylglucose (3-OMG) per centimeter intestinal length were greater in the active squirrels, but 3-OMG uptakes per milligram intestinal weight were greater in the hibernators, due to a significantly greater maximum rate of uptake (Jmax) per milligram with no differences in apparent Michaelis constant (Km). Passive permeabilities to L-glucose were similar in both groups. Total uptake of L-proline per centimeter was greater in active squirrels, but total proline uptake per milligram was greater in the hibernators due to a significantly greater Jmax per milligram with no difference in apparent Km. Na(+)-independent proline uptake accounted for a greater proportion of total proline uptake in active compared with hibernating squirrels. As a consequence, Na(+)-dependent proline uptake was greater in the hibernators when uptake was normalized either to intestinal length or intestinal weight. Thus hibernation is associated with an increase in the Jmax per milligram for 3-OMG and proline transport, as well as a shift in the Na+ dependency of proline uptake. We conclude that nutrient absorption is selectively retained in mammalian hibernators to maintain transport function after the extended winter fast.


Arterial baroreflex dynamics in normotensive and spontaneously hypertensive rats.

To investigate dynamic or frequency-dependent characteristics of arterial baroreflex control of efferent sympathetic nerve activity in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), we assessed the transfer function from aortic pressure (AP) to renal sympathetic nerve activity (RSNA) using a "white-noise technique." In pentobarbital sodium-anesthetized rats, we recorded RSNA as the output, while AP was randomly perturbed to impose input pressure changes with broad frequencies. We calculated the transfer function from AP to RSNA over the frequency range of 0.01-5 Hz through the spectral analysis of the input and output. The results indicated that the gain, phase shift, and coherence of the transfer function for SHR and for WKY were similar and statistically indistinguishable. The gain was relatively constant below 0.05 Hz but increased steadily by fivefold as frequency increased in the frequency range of 0.05-0.8 Hz. The phase was out of phase where coherence was high. The coherence was high (greater than 0.5) in the frequency range of 0.04-0.8 and 1.00-1.03 Hz but was low in other frequencies. These results suggest that dynamic or frequency-dependent characteristics of arterial baroreflex control of RSNA were not altered in SHR as compared with WKY.


Physiological concentrations of ANP exert a dual regulatory influence on renin release in conscious dogs.

The influence of physiological increments in circulating atrial natriuretic peptide (ANP) on renin release was determined in conscious dogs. Renin stimulus-response curves (RSRCs) were obtained by controlled reductions of renal perfusion pressure (RPP) under control conditions and during intrarenal or intravenous ANP infusions. Under all experimental conditions, the RSRCs were characterized by a plateau, a threshold pressure (Pth), and a steep slope below Pth. Intrarenal ANP infusion (0.9 ng.kg-1.min-1), which induced a calculated threefold elevation of renal arterial ANP concentration (but did not change systemic arterial ANP levels), increased the slope of the RSRC by 81% (P less than 0.05) with no effect on Pth. A quantitatively similar effect on the slope of the RSRC (+90%; P less than 0.05) was observed when systemic ANP levels were raised (from 37 +/- 2 to 71 +/- 9 pg/ml; P less than 0.05) by intravenous infusions (3.6 ng.kg-1.min-1). In addition, however, intravenously infused ANP reduced Pth from 91 to 85 mmHg (P less than 0.05), which caused a complete suppression of the renin response to a reduction of RPP down to 85 mmHg. These findings indicate that ANP can inhibit renin release at physiological plasma concentrations by shifting the RSRC to a lower pressure level; this shift is mediated by a modulation of extrarenal renin control mechanisms. The direct effect of ANP on renin release is one of stimulation.


Interactions of physiological and pharmacological concentrations of ANP and angiotensin II in conscious dogs.

This study in conscious, chronically instrumented dogs investigated the effects of human atrial natriuretic peptide [hANP-(99-126)] at physiological (5 ng.kg-1.min-1) and pharmacological (5-900 ng.kg-1.min-1) doses on angiotensin II (ANG II)-mediated effects on hemodynamics, renal excretion, and aldosterone release. Five female beagle dogs kept chronically on a dietary sodium intake of 2.5 mmol Na.kg body wt-1.day-1 received an intravenous infusion of 1, 4, 10, 20, and 50 ng.kg-1.min-1 ANG II (20-min periods) without (protocol 1) or with (protocol 2) simultaneous intravenous infusion of 5 ng.kg-1.min-1 hANP-(99-126). In protocol 1, glomerular filtration rate (means +/- SD) decreased from 4.0 +/- 0.6 to 2.8 +/- 0.5 ml.kg-1.min-1, renal sodium excretion (UNaV) decreased from 2.8 +/- 1.6 to 0.4 +/- 0.2 mumol.kg-1.min-1, and urine volume (V) decreased from 45 +/- 23 to 6 +/- 8 microliters.kg-1.min-1. There were no differences in the values between protocol 1 and protocol 2. Mean arterial blood pressure (MABP) increased similarly from 118 +/- 16 to 166 +/- 9 mmHg in protocol 1 and from 109 +/- 11 to 162 +/- 7 mmHg in protocol 2. Maximal aldosterone secretion was stimulated less in protocol 2 (481 +/- 92 vs. 362 +/- 158 pg/ml; P less than 0.05). In ANG II-pretreated (20 ng.kg-1.min-1) dogs (n = 4; protocol 3), intravenous hANP-(99-126) doses of 300-900 ng.kg-1.min-1 decreased MABP and central venous pressure.(ABSTRACT TRUNCATED AT 250 WORDS)


Hypothalamic and cortical sympathetic responses relay in the medulla of the rat.

Previously, investigations have indicated that the efferent pathway for sympathetic responses originating in the insular cortex (IC) must initially synapse in the lateral hypothalamic area (LHA). The LHA projects to both the ventrolateral medulla (VLM) and directly to the thoracic spinal cord. To determine the role of the VLM in mediating sympathetic responses from the IC and the LHA, in alpha-chloralose-anesthetized rats, renal nerve responses were recorded following electrical stimulation of these two forebrain sites before and after bilateral injection (300 nl) of cobalt (a synaptic blocking agent) into the VLM. The results demonstrated that a complete block of the increase in sympathetic nerve activity following stimulation of the IC or the LHA could be obtained with cobalt injections into the VLM. The most effective injection sites were located in the rostral ventrolateral medulla at the rostrocaudal middle and posterior regions of the C1 group of neurons. Chemical stimulation with DL-homocysteic acid, in the LHA, to activate cell bodies only evoked a decrease in arterial blood pressure and sympathetic nerve activity. These responses were also blocked by cobalt injection into the VLM. Injections of cobalt into the nucleus of the solitary tract did not block sympathetic responses elicited from the LHA. These results indicate that the efferent pathway for sympathetic responses from the IC through the LHA goes to the preganglionic region of the spinal cord via a mandatory synapse in the VLM.


Ca(2+)-related hepatocellular alterations during intra-abdominal sepsis.

Intra-abdominal sepsis was induced in rats by implanting into their abdominal cavities fecal-agar pellets impregnated with Escherichia coli and Bacteroides fragilis. Sham-operated rats received sterile pellets. A group of sterile- and septic-implanted rats was treated intraperitoneally with diltiazem (1.2 mg/kg) 8 h after implantations. Septic- and sterile-implanted rat hepatocytes were loaded with 1) the fluorescent dye indo-1 to quantify hepatocyte basal and vasopressin (100 nM)-elevated cytosolic Ca2+ concentration and 2) 45Ca to quantify Ca2+ flux and cellular content of exchangeable Ca2+. Lipid peroxidation was determined by measuring conjugated dienes (CD) and thiobarbiturate-reactive substances (TBA-RS) in liver homogenates. In septic-implanted rats, the basal cytosolic [Ca2+], cellular exchangeable Ca2+, Ca2+ flux, CD, and TBA-RS were significantly higher than in sterile-implanted rats. Although vasopressin caused a significant elevation in cytosolic [Ca2+] in septic rat hepatocytes, the magnitude of this elevation was significantly smaller than that found in the sterile group. Diltiazem treatment of septic rats significantly decreased basal cytosolic [Ca2+], cellular exchangeable Ca2+ content, Ca2+ flux, CD, and TBA-RS. Also, vasopressin-induced increase in hepatocyte cytosolic [Ca2+] in diltiazem-treated septic rats was significantly greater than that observed in untreated septic rats. Both Ca2+ and membrane lipid alterations were attenuated with diltiazem treatment of septic rats. These results suggest that prevention or attenuation of Ca2+ channel-mediated Ca2+ influx restores both Ca2+ homeostasis and membrane lipid alteration.


Forced oscillations in sympathetic nerve discharge.

Periodic electrical stimulation of the medullary raphe or lateral tegmental field in baroreceptor-denervated cats was used to force the central systems responsible for the 10-Hz and 2- to 6-Hz rhythms in post-ganglionic sympathetic nerve discharge (SND). The 10-Hz rhythm in SND could be entrained either to the frequency of medullary stimulation or to harmonics of the stimulus frequency. The harmonic of the stimulus frequency to which the 10-Hz rhythm was entrained in one postganglionic nerve could be different from that in another nerve. On this basis, we propose that the circuits responsible for the 10-Hz rhythms in SND may be modeled as a system of coupled nonlinear oscillators, each of which either influences one postganglionic nerve or nonuniformly affects different postganglionic nerves. The relatively wide band 2- to 6-Hz component in SND could be forced into a stable oscillatory state by medullary stimulation at frequencies between 3 and 5 Hz. This observation is consistent with the view that the 2- to 6-Hz component reflects the complex behavior of a nonlinear oscillator rather than the output of a physiological noise generator.


Low-dose near-celiac arterial cholecystokinin suppresses food intake in rats.

Exogenous cholecystokinin (CCK) suppresses food intake by acting on vagal sensory neurons. However, CCK doses used in behavioral experiments are generally much larger than those necessary to produce electrophysiological changes in vagal afferents. We made automated measurements of liquid food intake before, during, and after infusion of low doses of CCK octapeptide (CCK-8) through a chronic aortic catheter with its tip seated just above the celiac juncture. In parallel experiments, we made similar infusions while collecting blood from the hepatic portal and jugular veins for CCK assay. Injection of 10, 30, 50, and 70 pmol of CCK-8 suppressed feeding in a dose-dependent manner beginning 1 min postinfusion. The lowest dose to produce statistically significant suppression of preinfusion intake was 30 pmol. Infusion of the same CCK-8 doses into the jugular vein did not suppress feeding. Near-celiac injection of 30 pmol of CCK-8 produced systemic plasma CCK concentrations averaging 6.5 +/- 1 pM compared with less than 1 pM after saline injection. These findings show that exogenous CCK, by acting on tissues perfused by the celiac artery, can suppress feeding at doses that 1) are similar to those producing effects on the firing of vagal neurons and 2) do not increase plasma CCK concentrations above postprandial levels.


Placental glucose transport in heat-induced fetal growth retardation.

In six ewes heat stressed from 39 to 125 days gestation and studied in a normothermic environment at 135 days, fetal and placental masses were less than in control sheep (1,645 vs. 3,112 and 149 vs. 356 g, respectively, P less than 0.01). Umbilical glucose uptakes (Rf,UP) were measured keeping maternal arterial plasma glucose at 70 mg/dl at spontaneously occurring fetal plasma glucose values (state A) and at two additional fetal glucose levels, to determine the transplacental glucose difference (delta) vs. Rf,UP relation. At normal delta of 49.2 mg/dl, Rf,UP was less in the experimental group (3.2 vs. 5.6 mg.min-1.kg fetus-1, P less than 0.05). Differences in placental perfusion and glucose consumption could not account for this result, thus indicating a reduced placental glucose transport capacity. In state A, fetal hypoglycemia enlarged significantly (P less than 0.01) the delta to 56.7 mg/dl and increased Rf,UP approximately 50% over the Rf,UP at a normal delta. In heat-induced fetal growth retardation, fetal hypoglycemia increases the flux of maternal glucose across a placenta with reduced glucose transport capacity.


Muscle, joint, and tendon contributions to the torque profile of frog hip joint.

The relative contributions of muscle force, moment arm, and tendon compliance were determined as a function of joint angle in the frog semitendinosus-hip joint system. Muscle, joint, and tendon properties were individually measured and then combined to predict the torque generated at the hip joint as a function of joint angle (i.e., the hip torque profile). Predicted torques were then compared to experimentally measured torques using a stepwise regression model to quantify the relative importance of muscle, joint, and tendon contributions to the hip torque profile. Variation in moment arm accounted for 74% of the variability observed in the hip torque profile, while addition of the muscle's intrinsic sarcomere length-tension property accounted for an additional 19% of the torque profile variability. Tendon compliance, which permitted a small amount of sarcomere shortening, accounted for only about 4% of the torque profile variability. We conclude that in this muscle-joint system, the relative fiber length-to-moment arm ratio is the major determinant of the shape of the isometric joint profile. The fiber length-to-moment arm ratio in other mammalian systems is also discussed.


Both CCK-A and CCK-B/gastrin receptors are present on rabbit vagus nerve.

Cholecystokinin (CCK) receptors on vagal afferents have been implicated in many of the actions of the brain-gut peptide CCK, including satiety. Autoradiographic studies in rats have demonstrated the presence of CCK-A-type receptors on vagus nerves. However, direct and detailed characterization of this important CCK receptor site has never been reported with membrane-binding techniques. Using 125I-Bolt-on-Hunter-CCK octapeptide (125I-BH-CCK-8) and the recently discovered selective agonists and antagonists of CCK receptors, we have delineated the properties of CCK receptors on rabbit vagus nerve. 125I-BH-CCK-8 binding sites appeared to be homogeneous by the Scatchard analysis, with a dissociation constant of 0.14 nM and a maximum binding of 72 fmol/mg protein. However, competition studies using selective CCK ligands showed that the vagal CCK receptors are heterogeneous. A71378, a selective CCK-A agonist, showed biphasic displacement curves, with the high-affinity portion (less than 10 nM) accounting for approximately 60% and the low-affinity portion for approximately 40%. Competitive binding studies using A63387, a selective CCK-B/gastrin receptor agonist, also showed biphasic displacement curves, with the high-affinity portion (less than 30 nM) at approximately 40% and the low-affinity portion at approximately 60%. Under conditions which selectively examined vagal CCK-A or CCK-B/gastrin receptors, we demonstrated that a number of CCK subtype selective agonists and antagonists possessed similar affinities for the vagal CCK-A and -B/gastrin receptors as those found on the guinea pig pancreas (CCK-A) and cerebral cortex (CCK-B), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Effective loci and roles of acetylcholine in temperature regulation of goldfish.

Microinjections of acetylcholine (ACh) and carbachol were made into discrete forebrain loci in goldfish (Carassius auratus) to evaluate the importance of cholinergic mechanisms for behavioral thermoregulation. Injections of 5, 10, 25, and 50 micrograms ACh into the far anterior nucleus preopticus periventricularis (NPP) (R. Peter and V. Gill. J. Comp. Neurol. 159: 69-102, 1975) and immediately adjacent ventral telencephalon led to consistent dose-dependent decrease in selected temperature. No effect was observed following injections of 2 micrograms ACh or 0.7% NaCl. Injections of ACh into a different portion of the ventral telencephalon led to increases in the selected temperature. Lower doses of carbachol (0.5 and 1.0 micrograms) injected into the NPP produced decreases in selected temperature similar to the highest doses of ACh. Injections of ACh into loci other than those mentioned above either had no thermoregulatory effect or had lesser thermoregulatory effects which, in comparison with injections into the most effective sites, were inconsistent and required larger doses to obtain. The site where cholinergic stimulation led to decreases in the selected temperature exactly overlapped the effective site of ethanol hypothermia in the goldfish.


Development of blood pressure and cardiac reflexes in the frog Pseudis paradoxsus.

Systemic arterial blood pressure and heart rate (fH) were measured in unanesthetized, unrestrained larvae and adults of the paradoxical frog, Pseudis paradoxus from São Paulo State in Brazil. Four developmental groups were used, representing the complete transition from aquatic larvae to primarily air-breathing adults. fH (49-66 beats/min) was not significantly affected by development, whereas mean arterial blood pressure was strongly affected, being lowest in the stage 37-39 larvae (10 mmHg), intermediate in the stage 44-45 larvae (18 mmHg), and highest in the juveniles and adults (31 and 30 mmHg, respectively). Blood pressure was not significantly correlated with body mass, which was greatest in the youngest larvae and smallest in the juveniles. In the youngest larvae studied (stages 37-39), lung ventilation was infrequent, causing a slight decrease in arterial blood pressure but no change in heart rate. Lung ventilation was more frequent in stages 44-45 larvae and nearly continuous in juveniles and adults floating at the surface. Bradycardia during both forced and voluntary diving was observed in almost every advanced larva, juvenile, and adult but in only one of four young larvae. Developmentally related changes in blood pressure were not complete until metamorphosis, whereas diving bradycardia was present at an earlier stage.


Unique electrophysiological effects of dinitrophenol in Malpighian tubules.

In the course of electrophysiological studies of Malpighian tubules of the mosquito Aedes aegypti, we have found unusual effects of 2,4-dinitrophenol (DNP) that offer new insights into the electrogenic and conductive properties of the tubule. DNP (10(-4)M) depolarized the basolateral membrane voltage from -58.0 to -3.3 mV, and it depolarized the apical membrane voltage from 110.6 to 8.9 mV. In parallel the transepithelial electrical resistance increased from 11.4 to 16.8 k omega.cm, and the fractional resistance of the apical membrane increased from 0.32 to 0.57. On the assumption that measures of transepithelial resistance in the presence of DNP approach the shunt resistance, the experimental results indicate the following characteristics for the equivalent circuit of the tubule: 1) a shunt resistance that is approximately one-half the transcellular resistance, 2) low and high electromotive forces, respectively, at the basolateral and apical membranes of principal cells, 3) an electrogenic pump at the apical membrane, and 4) a basolateral membrane voltage that is due mostly to the voltage developed by current flow across the basolateral membrane resistance.


Ontogeny of fetal hepatic and placental growth and metabolism in sheep.

Ontogeny of fetal hepatic and placental growth and in vitro oxygen consumption (VO2) was investigated in fetal lambs at 75, 100, and 136 days postconception. Fetal hepatic relative weight and placental absolute and relative weights declined during this period. Oxygen consumption per gram dry weight of fetal liver and maternal placenta declined between mid and late gestation while fetal placental VO2 was unchanged. Estimated VO2 of the whole placenta did not change while the estimated total hepatic VO2 increased more than threefold between 75 and 136 days. Total hepatic VO2 was highly correlated with total placental VO2 at 136 days (r = 0.84). The results suggest that the placenta reaches its maximum growth and metabolic capacity before 100 days and possibly at or before midgestation. Changes in hepatic weight-specific total VO2, in addition to the declining relative size of the fetal liver, must contribute to the progressive decline in metabolic rate of the whole fetus during the second half of pregnancy. Correlations between placental and fetal liver weights and metabolic rates suggest the possibility of placental regulation of fetal hepatic growth and metabolism.


Coordination of respiration and swallowing: effect of bolus volume in normal adults.

The coordination of swallowing and respiration, as measured by nasal airflow, and the effect of changes in the volume of the swallow bolus (0-20 ml) were investigated in 12 normal subjects. Both nonbolus and bolus swallows were usually preceded and followed by expiratory airflow. Swallows followed by inspiratory airflow accounted for 20% of nonbolus swallows but decreased further in frequency in the presence of a bolus. Swallowing was associated with an apneic period lasting 1.90 +/- 0.26 s for nonbolus swallows. Based on the apneic period response to bolus volume, the subjects were divided into two groups. The apneic period decreased by 60% in seven of the subjects regardless of bolus volume. The remaining five subjects gradually increased swallow apnea as bolus volume was increased. At larger bolus volumes, the latter group also exhibited an earlier onset of the swallow apnea and an increase in the number of swallows preceded by inspiration. The duration of the swallow-associated respiratory cycle was similarly prolonged by an increase in bolus volume in both groups. The results indicate that the respiratory pattern associated with swallowing is modulated by the volume of the swallow bolus. Within the normal population, at least two different patterns of response to bolus volume are identified.


Arterial pressure lability and renal sympathetic nerve activity are dissociated in SAD rats.

The purpose of this study was to determine whether the sympathetic nervous system drives the high variability of arterial pressure (AP) observed after sinoaortic denervation (SAD) in rats. One or fourteen days after SAD, rats were instrumented chronically to record mean AP (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) in the conscious unrestrained state. Acute SAD increased MAP, HR, RSNA, and variability of MAP and decreased variability of both HR and RSNA. In rats with chronic SAD, variability of MAP remained high, whereas MAP, HR, RSNA, and variability of HR and RSNA returned to normal levels. Correlation analysis showed that, in sham-operated rats, AP and RSNA were negatively correlated in 90% of cases. In contrast, rats with both acute and chronic SAD exhibited only 30% negative and 25% positive correlations. These results indicate that 1) low AP variability in intact rats results from baroreflex-mediated inversely related fluctuations in RSNA and HR and 2) high variability of AP after acute and chronic SAD is correlated infrequently with RSNA. Because lability is reduced by interventions that block the sympathetic nervous system, we conclude that lability of AP associated with SAD appears to be mediated largely by a permissive role of sympathetic activity.


Effect of physical exercise in hypobaric conditions on atrial natriuretic peptide secretion.

To evaluate the role of atrial natriuretic peptide (ANP) in exercise-related cardiovascular and hormonal adjustments in hypobaric conditions, 14 young athletes performed a maximal ergometer test in a hypobaric chamber adjusted to simulate the altitudes of sea level and 3,000 m. Plasma immunoreactive ANP levels rose from 5.89 to 35.1 pmol/l at sea level and rose significantly less (P less than 0.05), from 5.36 to 22.3 pmol/l, at simulated 3,000 m. Plasma immunoreactive amino-terminal peptide of proANP (NT-proANP) levels increased to the same extent at sea level and at simulated 3,000 m (from 240 to 481 pmol/l and from 257 to 539 pmol/l, respectively). Plasma immunoreactive aldosterone increased significantly less at simulated 3,000 m (P less than 0.05), but the changes in plasma renin were similar in both conditions. Plasma immunoreactive endothelin-1 and serum erythropoietin levels remained unchanged. In conclusion, we found a blunted ANP response to maximal exercise of ANP in acute hypobaric exposure compared with that in normobaric conditions, but no significant difference in the NT-proANP responses between the two conditions. The divergence may be due to stimulation of the elimination mechanism of ANP.


Human IL-1 receptor antagonist partially suppresses LPS fever but not plasma levels of IL-6 in Fischer rats.

A human recombinant interleukin-1 receptor antagonist (IL-1ra) recognizes the two known IL-1 receptors and blocks the binding and many biological effects of both IL-1 alpha and IL-1 beta. The effectiveness of IL-1ra in modifying the fever and plasma IL-6 responses elicited by lipopolysaccharide (LPS) in vivo was tested in Fischer 344 rats. Animals that received IL-1ra 0.5 mg/kg intraperitoneally followed 10 min later by 10 micrograms/kg of LPS displayed significantly lower mean fever responses 2-4 h after injection than rats that received vehicle and LPS (0.48 +/- 0.13 vs. 0.95 +/- 0.16 degrees C, P = 0.016). Plasma levels of IL-6 at 4 h after injection were not different in IL-1ra-treated rats compared with controls (407,725 vs. 729,169 U/ml). Based on our previous finding that preadministration of antiserum to IL-1 beta markedly suppressed plasma IL-6 after LPS, and recent evidence that molar excesses of IL-1ra blocked IL-1-induced circulating IL-6 levels, the possibility that IL-1 is responsible for the induction of bioactive IL-6 during inflammation cannot be ruled out. Similarly, the inability of the IL-1ra to completely suppress the febrile responses of rats to LPS in the present study may be dose related. Alternatively, the induction of bioactive IL-6 by IL-1 in the rat may be mediated primarily through some receptor other than the type I (e.g., the type II receptor).


Behavioral and tissue responses to severe phosphorus depletion in cattle.

Two-year-old Aberdeen Angus heifers were depleted of phosphorus (P) by loss of phosphate in saliva from a parotid gland fistula combined with a low-P diet. The inorganic phosphate concentrations (Pi) of plasma, parotid saliva, ruminal fluid, and feces were reduced, but cerebrospinal fluid Pi was unaltered. Plasma Pi fell to less than 1.0 mM in 12 wk, and the cows displayed an avid appetite for old bones. P-deficient cows preferred old weathered bones to fresh bones until the latter had aged for approximately 1.5 yr. They did not eat blood, peritoneal fat, or meat, fresh or aged for 2-3 yr. They did not eat Na or Ca phosphate salts or bone heated to greater than 250 degrees C. P-replete cows did not take or eat bones. The behavioral effects of P deficiency were associated with failure to gain body weight and maintain condition, significant bone changes indicated by reduced bone weight and specific gravity, osteopenia indicated by radiology, evidence of reduced bone formation indicated by plasma osteocalcin levels and histology, and abnormal estrous cycles. Prolonged P deficiency was associated with increased plasma concentrations of total Ca and 1,25-dihydroxyvitamin D and reduced plasma concentrations of parathyroid hormone.


Hypoxia attenuates the renin response to hemorrhage.

We studied hypoxia and hypotensive hemorrhage in conscious female goats. After control, goats continued an experimental period in normoxia or hypoxia [fractional inspired oxygen concentration (FIO2) = 0.10] for 120 min. After 60 min in the experimental period, a hemorrhage (0.5 ml.kg-1.min-1 for 30 min) was initiated (normoxic hemorrhage, NH; hypoxic hemorrhage, HH). Heart rate (HR) increased 51 +/- 18 beats/min with NH after 30 min of hemorrhage. HR increased 40 +/- 10 beats/min after hypoxic gas introduction, with no further increase during HH. Mean arterial blood pressure (MABP) was reduced 23 +/- 7 mmHg 30 min after completion of blood loss with normoxia but was reduced 23 +/- 7 mmHg at 20 min of HH. Arginine vasopressin (AVP) was increased to 2.60 +/- 2.08 and 160.40 +/- 49.74 microU/ml after 10 and 20 min of HH, respectively, and was only increased after 30 min (87.33 +/- 67.18 microU/ml) of NH. Unexpectedly, plasma renin activity (PRA) increased in parallel in both groups and was doubled at 30 min of hemorrhage. Atrial natriuretic factor was reduced to 8.8 +/- 1.6 pg/ml by 10 min of NH and to 11.4 +/- 3.3 pg/ml at 30 min of HH. Thus hypoxia leads to an earlier development of hypotension and increase in AVP with blood loss but may attenuate the PRA response to blood pressure reduction.


Regional interactions between thermosensitive neurons in diencephalic slices.

Rat brain slices were used to investigate regional interactions between thermosensitive neurons in different diencephalic regions. Horizontal tissue slices rested over three thermodes. This permitted independent thermal stimulation of rostral, middle, and caudal regions. Thermocouples measured tissue temperatures in these three locations, and extracellular recordings measured neuronal responses to temperature changes both locally (at the site of the recorded neuron) and in remote regions of the slice. Many of the neurons that were sensitive to remote temperatures were located near the lateral border of the diencephalic nuclei, especially in the perifornical area. All neurons displaying remote thermosensitivity also displayed local thermosensitivity. These neurons usually showed opposite responses to remote and local temperatures; i.e., most of these neurons were locally warm sensitive but showed cold sensitivity to remote temperatures. These findings indicate that thermosensitive synaptic networks extend throughout the diencephalon and may explain the effect of temperature on a variety of homeostatic systems.


Delayed firing rate responses to temperature in diencephalic slices.

Thermoregulatory responses may be delayed in onset and offset by several minutes after changes in hypothalamic temperature. Our preceding study found neurons that displayed delayed firing rate responses during clamped thermal stimulation in remote regions of rat diencephalic tissue slices. The present study looked for similar delayed firing rate responses during clamped (1.5-10 min) changes in each neuron's local temperature. Of 26 neurons tested with clamped thermal stimulation, six (i.e., 23%) showed delayed responses, with on-latencies of 1.0-7.8 min. These neurons rarely showed off-latencies, and the delayed response was not eliminated by synaptic blockade. The on-latencies and ranges of local thermosensitivity were similar to delayed neuronal responses to remote temperature; however, remote-sensitive neurons displayed off-latencies, higher firing rates at 37 degrees C, and greater sensitivity to thermal stimulation. Our findings suggest that delayed thermosensitivity is an intrinsic property of certain neurons and may initiate more elaborate or prolonged delayed responses in synaptically connected diencephalic networks. These networks could explain the delayed thermoregulatory responses observed during hypothalamic thermal stimulation.


Determining energy expenditure in preterm infants: comparison of 2H(2)18O method and indirect calorimetry.

The doubly labeled water (2H(2)18O) method used to estimate total energy expenditure (EETotal) is particularly sensitive to analytic error in preterm infants, because of their high percentage of body water and the high ratio of water flux to CO2 production. To evaluate further use of this method, the EE of 12 preterm infants was measured by indirect calorimetry and 2H(2)18O simultaneously and continuously for 5 days. Initial infant weight, age, and postconceptional age were (means +/- SD) 1,674 +/- 173 g, 4.4 +/- 2.6 wk, and 34.6 +/- 1.6 wk, respectively. The indirect calorimeter system included an air-temperature-controlled chamber and heart rate monitor. EE was measured by indirect calorimetry for 85.6 +/- 4.7% of study time and estimated from the linear regression of heart rate on EE for 14.4 +/- 4.7% of study time. The 2H(2)18O method entailed an initial dose of 100 mg 2H2O and 250 mg 18O/kg and a final dose of 75 mg 18O/kg; urine was collected twice daily. 2H and 18O enrichments were measured by gas-isotope-ratio mass spectrometry. EE was calculated from measured 2H and 18O dilution spaces (NH, NO), turnover rates (kH, kO), and measured respiratory quotient. The ratio of 2H to 18O dilution spaces was 1.01 +/- 0.01 and the ratio of kO to kH was 1.16 +/- 0.04. Estimation of EE from 2H(2)18O and indirect calorimetry agreed within 1%, although individual variability in methods was large.


Baroreflex control of arterial blood pressure during involuntary diving in ducks (Anas platyrhynchos var.).

The dynamic role of arterial baroreceptors in control of mean arterial blood pressure (MAP), heart rate (HR), cardiac output (CO), hindlimb vascular (HLVR) and total peripheral (TPR) resistance responses to forced dives was investigated in acutely and chronically barodenervated ducks. To activate the baroreflex, the proximal end of one aortic nerve was stimulated electrically with bipolar electrodes that had been implanted under pentobarbital sodium anesthesia. Predive nerve stimulation caused CO to fall (by reducing HR; stroke volume remained constant), producing a decrease in MAP to half the prestimulation level. During diving (for 2.5-min periods) nerve stimulation did not affect HR and MAP after the first minute of submersion. Neither HLVR nor TPR contributed to the fall in MAP during aortic nerve stimulation before or during diving. The effects of nerve stimulation on HR and MAP were maintained to the end of dives in animals given 100% O2 to breathe before diving. In separate experiments, increasing arterial chemoreceptor input by perfusing one vascularly isolated carotid body with venous blood caused a reduction in the effects of aortic nerve stimulation on MAP. Arterial baroreceptors may thus act on HR to alter MAP early in the dive, but as the dive progresses the baroreflex is attenuated by an increase in peripheral chemoreceptor drive.


Tumor necrosis factor-beta induces sleep, fever, and anorexia.

The enhanced sleep, fever, and anorexia experienced during general infections are attributed to the increased production of cytokines. Cytokines such as interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) have characteristic somnogenic, pyrogenic, and anorectic effects. TNF-beta is closely related to TNF-alpha, and they share common receptors. The effects of TNF-beta on sleep-wake activity, brain temperature (Tbr), and food intake were, however, heretofore unknown. We injected 0.5-200 ng TNF-beta into rabbits intracerebroventricularly (icv) in the light period, and the electroencephalogram, movement, and Tbr were recorded for 6 h from rabbits. The highest dose, 200 ng TNF-beta, induced increases in non-rapid-eye-movement sleep and decreases in rapid-eye-movement sleep accompanied with biphasic febrile responses. Icv injection of 100 ng TNF-beta at dark onset suppressed 12-h and 24-h food intake in rats. These data suggest to us that TNF-beta may belong to the group of endogenous pyrogens/sleep factors.


Selective activation of norepinephrine- and epinephrine-secreting chromaffin cells in rat adrenal medulla.

The differential effects of insulin-induced hypoglycemia and cold exposure on adrenal medullary epinephrine (Epi) and norepinephrine (NE) cells were investigated in male Sprague-Dawley rats. In rats fasted overnight, insulin produced a marked hypoglycemia that resulted in a 70% decrease in adrenal medullary Epi content 3 h after the insulin was administered. No change in NE content was observed. Plasma Epi concentration was increased markedly after insulin, with a smaller increment in NE. In contrast, exposure to a 4 degrees C environment selectively reduced adrenal NE content, with the effect reaching statistical significance at 18 h. Cold exposure also led to a significant rise in plasma NE but not Epi. Both insulin-induced hypoglycemia and cold exposure significantly elevated adrenal dopamine, indicating that catecholamine synthesis was stimulated. Further evidence of enhanced catecholamine formation was the observation that inhibition of synthesis with alpha-methyl-p-tyrosine (AMT) greatly augmented the ability of insulin-induced hypoglycemia to selectively reduce adrenal medullary Epi content. Similarly, in cold-exposed animals, AMT pretreatment accelerated the NE depletion so that a significant decline was observed at 3 h. These results support the conclusion that the two major populations of adrenal catecholamine-secreting cells may be preferentially stimulated by different stressors. Moreover, augmented synthetic activity functions to maintain catecholamine stores in both Epi- and NE-secreting cells.


Habituation of lactate release responding to stressful stimuli in rat prefrontal cortex in vivo.

The extracellular level of lactate is mainly dependent on functional glucose metabolism in most brain cells regulated in part by neuronal activity. We investigated the effect of environmental stimuli on lactate release in rat medial prefrontal cortex (MPFC) by in vivo brain microdialysis. Lactate release was clearly demonstrated to increase immediately and transiently from its steady-state levels after various stimuli (tail pinch for 1 min, 100-dB white noise for 5 min, and immobilization for 5 min). These stimuli-induced increments were depressed after a second trial 1 h later in a manner consistent with dishabituation to a novel stimulus and "below-zero" habituation by several repetition. Likewise the local perfusion of dopamine or norepinephrine transiently increased and then diminished the second perfusion-induced increase in MPFC lactate release consistent with desensitization. These results suggest the possibility that stressful stimuli produce one-trial habituation of lactate release in MPFC via a mechanism involving the regulation of the catecholaminergic system.


Microsphere and dilution techniques for the determination of blood flows and volumes in conscious mice.

Although the mouse is the most commonly used transgenic species, little is known regarding cardiovascular and fluid homeostasis in this animal. Therefore, the reference microsphere and dilution techniques were adapted for the measurement of cardiac output (CO), regional blood flows, and intravascular fluid volumes in the conscious mouse. Previously acclimatized C3H mice were studied 4-5 h after surgery and recovery from anesthesia. Approximately 40,000 85Sr-labeled microspheres were injected into the left ventricle while a reference sample was withdrawn at one of two rates from the femoral artery. 51Cr and 125I were used for the determination of blood volume (BV), plasma volume (PV), and Fcells ratio (whole body hematocrit/large vessel hematocrit). CO and BV in the conscious mouse were 16 +/- 1.4 ml/min and 2.3 +/- 0.1 ml, respectively. Anesthesia lowered heart rate, blood pressure, PV, and altered the distribution of CO. Two successive injections of 15,000-20,000 microspheres were tolerated in the mouse without an increase in total peripheral resistance. The results indicate that the microsphere and indicator dilution techniques can be applied to study cardiovascular and fluid homeostasis in the mouse.


Alterations in oxytocin prohormone processing during early development in the fetal sheep.

Oxytocin (OT) prohormone processing was studied in fetal sheep. Using specific antisera that recognize the amidated and the COOH-terminal extended forms of OT, we measured arterial and venous levels of the OT peptides in fetal sheep plasma at 94 and 138 days of gestation. Plasma levels of the COOH-terminal extended forms, OT-X, were highest early in development, 35.7 +/- 9.8 vs. 14.3 +/- 5.7 pg/ml (94 vs. 138 days). The ratio of the plasma peptides, OT-X to OT, was higher in the young fetus (35 +/- 11.6 vs. 3.1 +/- 1.3, 94 vs. 138 days). There were also developmental changes in the umbilical artery-umbilical vein differences, with positive values noted in late gestation. These results demonstrate that the changes in the processing of the OT precursor that occur during fetal development are reflected by alterations in the relative amounts of prohormone and amidated hormone found in fetal plasma.


Regulation of apical Cl- conductance and basolateral K+ conductances by phorbol esters in HT-29cl.19A cells.

The effect of phorbol esters [4 beta-phorbol 12,13-dibutyrate (PDB) and phorbol 12-myristate 13-acetate (PMA)] on potential differences and resistances was studied with the conventional microelectrode technique applied to confluent filter-grown monolayers of the human colon carcinoma cell line HT-29cl.19A. Phorbol esters (PDB or PMA from 10(-7) to 10(-6) M) evoked 1) a transient increase in the transepithelial potential difference (peak value 3.5 +/- 0.5 mV), 2) a depolarization of the cell potential by 23 +/- 2 mV at the peak of the transepithelial potential change and a continued decrease during the decline of the transepithelial potential, and 3) a decrease of the fractional resistance of the apical membrane consisting of two phases, a relative rapid one (time constant 1.2 +/- 0.2 min) and a much slower further decrease during the second phase (time constant 34 +/- 1 min). Ion replacements and electrical circuit analyses indicate that PDB activates an apical Cl- conductance and slowly inhibits the basal K+ conductance of the basolateral membrane. PDB reduced the transepithelial response to forskolin due to inhibition of the basal K+ conductance. The Ca2+ ionophore ionomycin accelerated that effect of PDB. Staurosporine inhibited the effects of PDB, suggesting that the PDB effects are mediated via activation of a protein kinase C.


Novel bumetanide-sensitive K+ transport in preimplantation mouse conceptuses.

Ouabain-resistant K+ transport activity was characterized primarily by measuring Rb+ uptake because 86Rb+ has a more convenient half-life than 42K+. Ouabain-resistant 86Rb+ uptake by mouse two-cell conceptuses and blastocysts was slowed by the K(+)-Na(+)-2Cl- cotransporter inhibitors bumetanide [inhibitory constant (Ki) = 400 nM] and furosemide (Ki approximately 10 microM), but it was insensitive to a variety of K+ channel blockers. This component of 86Rb+ transport was also inhibited by K+ and nonradioactive Rb+ and it was stimulated by Cl-. Nevertheless, neither 36Cl- nor 22Na+ uptake was inhibited by bumetanide, whereas 42K+ uptake was inhibited by both bumetanide and furosemide. Bumetanide-sensitive Rb+ transport in blastocysts had a Hill coefficient of 1.0 and a Michaelis constant value of 3.0 mM. By these criteria, preimplantation conceptuses contain a novel, bumetanide-sensitive K+ transport system that does not cotransport Cl- or Na+. Moreover, bumetanide-sensitive Rb+ uptake was 10 times faster in blastocysts when they were collapsed to expose the basal membrane of the trophectoderm to 86Rb+ in the medium. Therefore, the novel system may be located predominantly in the basal rather than in the apical membrane of the trophectoderm.


Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study.

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.


Muscle fatigue in the frog semitendinosus: role of the high-energy phosphates and Pi.

The purpose of this study was to determine the concentration of ATP, phosphocreatine (PC), Pi, lactate, and glycogen in single frog skeletal muscle fibers and assess their role in the etiology of muscle fatigue. The frog semitendinosus (ST) muscle was fatigued, quick frozen at selected time points of recovery, and freeze-dried, and single fibers were dissected, weighed, and assayed for ATP, PC, lactate, Pi, and glycogen. The fatigue protocol reduced peak tetanic force (Po) to 8.5% of initial, while ATP and PC decreased from 45.18 to 33.16 and 128.90 to 28.76 mmol/kg dry wt, respectively. Lactate and Pi increased from 29.36 to 100.84 and 33.04 to 142.50 mmol/kg dry wt, respectively. It is doubtful that the small decline in ATP limited cross-bridge force production. Although a significant correlation between the recovery of PC and Po was demonstrated (r = 0.994), the time period showing the fastest rate of force recovery coincided with little change in PC. A significant correlation was demonstrated between the recovery of both total and the H2PO4- form of Pi and Po. In conclusion, the results of this study are incompatible with the hypothesis that the high-energy phosphates (ATP and PC) mediate muscle fatigue. The large increase in Pi with stimulation and the high correlation between the recovery of both total and the H2PO4- form of Pi and Po support a role for Pi in the production of skeletal muscle fatigue.


Intracardiac detection of angiotensinogen and renin: a localized renin-angiotensin system in neonatal rat heart.

There is increasing evidence that the renin-angiotensin system (RAS) modulates cardiovascular function through both blood-borne and tissue-derived components. The existence of a local RAS has been proposed in the heart based on biochemical and molecular biological studies that identify angiotensinogen and renin. We conducted the present study to determine the chamber localization of angiotensinogen and renin mRNA in neonatal rat heart and whether these components could be identified in cultured cardiomyocytes and fibroblasts obtained from neonatal rat heart. Experiments using polymerase chain reaction (PCR) indicated that whole hearts obtained from neonatal rats contained both angiotensinogen and renin mRNA. With the use of radiolabeled cDNA probes and in situ hybridization, angiotensinogen and renin transcripts were localized both in the atria and ventricles of neonatal rat hearts. Relative signal strengths for angiotensinogen were highest in the left and right ventricles. In contrast, renin signal strength was overall much lower and preferentially localized in the left ventricle. To investigate the cellular source of angiotensinogen and renin, cultured neonatal heart cardiomyocytes and ventricular fibroblasts were screened for angiotensinogen and renin messenger RNA and protein using PCR and indirect immunofluorescent staining, respectively. These experiments demonstrated that both cell types produce transcripts and the respective translation products for angiotensinogen and renin. These data suggest that the site of angiotensin II synthesis can occur at the level of the individual cardiomyocyte and fibroblast, where it may serve to directly and/or indirectly regulate cardiac rate, force, growth, and development in the neonate.


Differential megakaryocytic desensitization to platelet agonists.

Platelets are released into the peripheral circulation from the bone marrow where they arise as fragments of megakaryocyte cytoplasm. To characterize the effects of platelet agonists on megakaryocytes, we examined calcium signaling and desensitization to thrombin, the thromboxane A2 (TxA2) mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), and platelet-activating factor (PAF) in cultured CHRF-288-11 megakaryocytic cells. Initially, we compared agonist-stimulated calcium transients in fura-2-loaded CHRF-288-11 cells and human platelets. The 50% effective concentration values for the agonists to increase free cytosolic calcium were as follows: thrombin (0.11 +/- 0.02 U/ml in CHRF, 0.19 +/- 0.03 U/ml in platelets), U46619 (147 +/- 33 nM in CHRF, 157 +/- 5 nM in platelets), and PAF [15 +/- 2 nM in CHRF, 16 +/- 2 nM in platelets (n = 4 each)]. CHRF-288-11 thrombin, TxA2, and PAF receptors were demonstrated to be coupled to phospholipase C because each of the agonists stimulated phosphatidylinositol hydrolysis in myo-[3H]inositol-loaded CHRF-288-11 cells and pharmacological inhibition of phospholipase C-blunted agonist-stimulated calcium signaling. CHRF-288-11 cells exposed to the three agonists for 1 h showed different patterns and extent of homologous and heterologous desensitization. Protein kinase C activation appeared to be necessary but not sufficient for desensitization because 1) activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited the calcium responses to all three agonists, 2) inhibition of protein kinase C with staurosporine attenuated subsequent desensitization to each agonist, and 3) each agonist increased protein kinase C activity in CHRF-288-11 cell homogenates.


Cellular mechanisms of vasopressin and endothelin to mobilize [Mg2+]i in vascular smooth muscle cells.

The present study was undertaken to examine the effects of arginine vasopressin (AVP) and endothelin-1 (ET-1) on cytosolic free Mg2+ ([Mg2+]i) in cultured rat vascular smooth muscle cells (VSMC). [Mg2+]i was measured using the fluorescence indicator dye mag-fura-2. AVP and ET-1 at a concentration of 1 x 10(-9) M or higher induced the mobilization of [Mg2+]i and cytosolic free Ca2+ ([Ca2+]i) in a dose-dependent manner in rat VSMC. Atrial natriuretic peptide and sodium nitroprusside producing cellular guanosine 3',5'-cyclic monophosphate did not affect [Mg2+]i and [Ca2+]i. A diterpene activator of adenylate cyclase, forskolin, also did not alter [Mg2+]i and [Ca2+]i. The removal of extracellular Mg2+ enhanced the AVP-mobilized [Ca2+]i and did not change the AVP-mobilized [Mg2+]i. The Ca(2+)-free and nominally Mg2+/Ca(2+)-free states decreased the AVP-mobilized [Mg2+]i and [Ca2+]i. The Na(+)-free state enhanced the sustained, but not peak, level of the AVP-mobilized [Mg2+]i. These results indicate that AVP and ET-1 mobilize [Mg2+]i mediated through their intracellular second messenger [Ca2+]i and independent of extracellular Mg2+. Also, an increase in [Mg2+]i is indicated to stimulate the Na(+)-Mg2+ exchange to increase cellular Mg2+ efflux.


Immunolocalization of chloride-transporting membrane vesicles in tracheal epithelial cells.

A membrane fraction eluted from a phenyl Sepharose column (MPS) was isolated from renal cortex and bovine tracheal epithelia that is enriched in a single type of Cl- channel [C. L. Preston, M. A. Calenzo, and W. P. Dubinsky. Am. J. Physiol. 263 (Cell Physiol. 32): C879-C887, 1992]. A 200-kDa membrane protein that copurifies with and appears to be specific to this fraction was purified and used to raise antisera for immunological characterization of these membranes. The antisera reacted in immunoblots with a 200-kDa protein in homogenates of bovine trachea, kidney, pancreas, lung, and intestine. There was also cross-reactivity with a 200-kDa protein in immunoblots in rat stomach, pancreas, and lung. There was no cross-reaction with rat skeletal muscle, cardiac muscle, or aorta. Thus this protein appears to be preferentially enriched in epithelial tissues. Examination of each major fraction during the purification of MPS membranes from trachea shows no enrichment of the 200-kDa protein in plasma, mitochondrial, or nuclear membrane fractions. The only significant enrichment was observed in MPS that is purified by hydrophobic chromatography. In frozen sections, antisera and monospecific immunoaffinity-purified antibodies localize the protein primarily to the apical domain of tracheal columnar epithelial cells with small punctate structures throughout the cytoplasmic compartment.


Lysophosphatidic acid induces a pertussis toxin-sensitive Ca(2+)-activated Cl- current in Xenopus laevis oocytes.

Lysophosphatidic acid (LPA) induces a Ca(2+)-activated Cl- current in defolliculated Xenopus laevis oocytes. The response appears mediated by a specific membrane receptor, because no current is induced when related compounds [phosphatidic acid (PA), lysophosphatidylcholine (LPC), and lysophosphatidylserine (LPS)] are applied extracellularly or when LPA is injected intracellularly. Incubation in pertussis toxin prevents the response. The response is mediated by a Ca(2+)-activated Cl- current because 1) it is abolished by intracellular ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 5 mM) but not affected by changes in extracellular Ca2+ concentration and 2) the reversal potential becomes more positive at lower Cl- concentrations. Suramin (2 mM) blocks the LPA-induced current, but PA, LPS, LPC, and the platelet-activating factor antagonist WEB-2086 do not. The response is dose dependent for LPA concentrations from 10(-8) to 10(-3) M. Incubation of oocytes in LPA does not induce germinal vesicle breakdown. These findings suggest that this novel oocyte response to LPA is mediated by a specific membrane receptor linked to a pertussis toxin-sensitive G protein.


Organic osmolytes increase cytoplasmic viscosity in kidney cells.

The hypothesis was tested that accumulation of osmolytes by kidney cells grown in hyperosmolar media decreases the rotational and translational mobilities of small polar solutes in the cytosolic compartment. Rotational mobility was measured by the picosecond rotational correlation times (tau c) of 2',7'-bis(2-carboxyethyl)-5(6)carboxylfluorescein (BCECF) by multiharmonic microfluorimetry. In isolated segments of rabbit proximal tubule, thick ascending limb, and cortical collecting duct that were perfused and bathed in 300 mosM media, tau c were in the range 180-250 ps, corresponding to apparent rotational viscosities (eta r) of 1.1-1.5 cP. In cortical collecting tubule, eta r was not influenced by serosal vasopressin. In Madin-Darby canine kidney (MDCK) cells grown in 300-1,200 mosM media, eta r increased progressively by up to a factor of 1.38 +/- 0.03; measurements of tau c and macroscopic viscosity in artificial solutions containing osmolytes supported the hypothesis that the increased eta r was due to accumulation of organic osmolytes. BCECF translational mobility was measured by fluorescence photobleaching recovery using a focused 1.2-microns diameter Ar laser beam at 488 nm. Recovery half-times were 36 +/- 3 (SE) ms (n = 10) in MDCK cells grown in 300 mosM media and 62 +/- 3 ms (n = 10) when grown in 1,200 mosM media. The results suggest that accumulation of osmolytes by renal cells is associated with significantly increased cytosolic viscosity. The increased viscosity would slow enzymatic and transport processes in the cytosolic compartment.