Changes in the jugular haematocrit of sheep during feeding.

Intact and splenectomized sheep with and without a rumen fistula were used to investigate changes in the jugular blood haematocrit and plasma osmolality during hourly and once-daily feeding regimes. Osmolality was also estimated in the ruminal fluid of fistulated sheep with spleens. Haematocrit decreased in sheep with spleens before they were given a once-daily feed; it increased when these sheep started to feed, reaching a maximum increase of 13% after 30 min of feeding; it decreased during the remaining 45 min of feeding time and usually continued to decrease after feeding stopped. These changes were not due to diurnal influences. Splenectomized sheep fed once daily showed only small decreases in haematocrit before they were fed. Increases occurred with the onset of eating but they were smaller (7%) than in intact sheep and were of shorter duration. In hourly fed sheep with spleens, haematocrit decreased in the early stages of sampling in a manner similar to that for sheep fed once daily. The changes in haematocrit that did occur were not related in any obvious manner to the feeding regime. The haematocrit in splenectomized sheep fed hourly was stable throughout feeding. Variations in the haematocrit in splenectomized sheep, equivalent to a range of 13% in one of them, were observed in a series of blood samples obtained during a 5-h period remote from the feeding time. Large increases occurred in osmolality of ruminal fluid when sheep were fed daily and this was abolished by hourly feeding. Plasma osmolality in sheep fed once daily increased slowly. Maxima occurred after 100 min from the start of eating and were 7% greater than prefeeding values. Only minor changes were observed when these sheep were fed hourly.     

Changes in arterial blood pressure, heart rate and haematocrit during acute hyperkalaemia in conscious sheep.

The systolic, diastolic and mean arterial blood pressures, heart rate and haematocrit were measured at 15 minute intervals before, during and after 2 hour infusions of 0-4 mol.l-1 NaCl at 2-2 ml min-1 into conscious intact sheep and 0-4 mol. l-1 KCl at 2-2 ml. min-1 into conscious sheep which were either intact or adrenalectomized. The haemotocrit was also measured in splenectomized sheep receiving 0-4 mol. l-1 KCl. The NaCl infusion had no significant effect on blood pressure(BP), heart rate and haematocrit. Both intact and adrenalectomized sheep were able to withstand an increase in plasma potassium concentration in excess of 50% of the preinfusion concentration before any substantial fall in BP occurred. In intact and adrenalectomized sheep, heart rate and haematocrit increased rapidly and progressively throughout the potassium infusions and at maximum plasma potassium concentration the mean increments in these parameters for both groups of sheep were 21-6+/-2-69 beats/min and 7-5+/-0-47% respectively. Heart rate and haematocrit were more closely correlated with the plasma potassium concentration than with any other variable measured in these experiments. Adrenalectomy did not reduce the ability of the sheep to maintain their BP or to increase their heart rate and haematocrit. As the mean increase in haematocrit during potassium infusion into splenectomized sheep was 1-3+/-0-45% most of the increase in haematocrit observed in the potassium-infused intact and adrenalectomized sheep was caused by ejection of red cells from the spleen into the circulation.     

Plasma insulin concentrations during infusions of potassium and sodium chloride solutions into conscious splenectomized sheep

Haematocrit values, plasma osmolality and the plasma concentrations of sodium, potassium, chloride and insulin were measured in carotid arterial blood before, during and after intravenous infusion of NaCl (0.5 mol 1-1) and KCl (0.5 mol 1-1) at 2 ml min-1 for 105 min into six conscious splenectomized sheep. Hypertonic NaCl infusion was associated with a fall in haematocrit of 1.30 +/- 0.10% (P less than 0.001) and no consistent change in plasma insulin concentration occurred during this infusion. Hypertonic KCl infusion caused the haematocrit to increase by 1.70 +/- 0.39% (P less than 0.001) and the plasma insulin concentration to increase by 60.0 +/- 16.3 mu U ml-1 (P less than 0.01). It was concluded that this increase in insulin concentration was caused by elevation of the plasma potassium concentration and was not due to coincident increases in plasma chloride concentration or osmolality. Shrinkage of the extracellular fluid volume during KCl infusion made no major contribution to the increase in insulin concentration which was probably the result of increased release from the pancreas.     

Role of spleen in ANF-induced reduction in plasma volume.

Atrial natriuretic factor (ANF) causes an increase in hematocrit that cannot be accounted for by urinary losses. The mechanism behind this phenomenon was studied in intact and splenectomized rats. Rat ANF 99-126 was infused i.v. for 30 min into conscious rats at rates of 0 (saline control), 0.05, or 0.1 microgram/min. Plasma volume was then determined by dilution of the dye, Evan's Blue. In one group of rats, red cell volume was determined using 51Cr-labelled erythrocytes. ANF infusion was continued uninterrupted throughout the experiments. In the intact rats, ANF (0.10 microgram/min) caused hematocrit to increase from 38.9 +/- 0.5 to 41.2 +/- 0.4% (p < 0.005). Splenectomy so attenuated this response to ANF that it failed to reach significance. Similarly, ANF (0.10 microgram/min) caused plasma volume to fall from 5.1 +/- 0.1 to 4.5 +/- 0.1 mL/100 g body wt. (p < 0.005) in the intact rats, but did not affect plasma volume in the splenectomized rats. As a result, blood volume was significantly reduced by ANF in the intact rats, but remained unchanged in the splenectomized rats. Red cell volume did not change in response to infusion of ANF, nor did ANF affect the rate of clearance of Evan's Blue out of the plasma. It is concluded that the spleen is an important site of movement of protein-poor fluid out of the vasculature, and that this exchange is influenced by ANF.     

The role of blood coagulability and axial streaming of erythrocytes in determining F cells-value and TPR.

Experiments were performed in anaesthetized and splenectomized dogs to influence the intravascular distribution of erythrocytes and plasma within the vascular bed. Blood volume was determined by double-isotope labelling of red cells and plasma. A significant decrease of F cells-value was found after posthaemorrhagic haemodilution and after haemodilution induced by homologous plasma. The administration of epsilon-amino caproic acid and bovine thrombin was followed by a significant increase of F cells-value. An interdependence of coagulability and F cells-value is suggested. The increase of haematocrit was followed by an increase of TPR, which, however, was only half of the value that would have been anticipated on the basis of the increase of viscosity when measured in normal-bore viscometers. This points to the possibility that blood flowing in the resistance vessels undergoes a certain spontaneous separation of cells and plasma and its haematocrit will be lower than in the large vessels. Cell separation in different organs having different haematocrit values and blood volumes probably affects total body haematocrit and F cells-value.     

Effects of insulin hypoglycaemia in the sheep on jugular haematocrit and plasma corticosteroid concentrations.

Insulin hypoglycaemia, by acting as a stressor, caused an increase in plasma corticosteroid concentration in sheep. It did not increase jugular haematocrit in splenectomized sheep, but caused an increase, presumably by splenic contraction, in the following sheep: two control, one with one adrenal cortex as its only adrenal tissue, two with denervated spleens, and two splanchnicotomized animals. These preparations showed that insulin hypoglycaemia can cause a splenic contraction in the absence of an increase in plasma adrenaline and after splenic extrinsic denervation.     

Splenic storage volume in the unanesthetized resting beagle

Determination of the dog's splenic storage volume without surgical procedures requires measurement of both circulating and total red cell volumes. The estimation of circulating red cell volume by radioisotope techniques is impeded by the rapid uptake of tagged cells in the spleen. The circulating cell volume might be calculated from plasma volume and large vessel hematocrit, provided that the latter is corrected for the unequal distribution of red cells in the circulatory system. However, the correction factor can only be estimated in the splenectomized dog. We describe here a method to determine the factor in the intact dog, "physiologically splenectomized" by the severe exercise. The values obtained by this method slightly exceed those in the resting dog, as shown by studies in splenectomized exercising beagles in which splenic function was simulated by infusion of packed cells. The method was tested in beagles exercised by swimming and treadmill running and it was concluded that in the unanesthetized resting beagle about one-third of all erythrocytes is stored in the spleen. Labeled cells are equilibrated with about one-half of the splenic storage volume within 10 min after their injection. During maximal exertion the mean increase in large vessel hematocrit was 38.6 +/- 3.3%, the mean decrease in plasma volume 13.6 +/- 1.7% and the mean increase in plasma osmolarity 2.8 +/- 0.9% (percentages of control values).     

Plasma volume and proteins in voluntary hyperventilation

In a controlled study the changes of the plasma volume and plasma proteins induced by voluntary hyperventilation (HV) were investigated in nine splenectomized volunteers. The plasma volume changes were calculated from the changes of the hemoglobin and hematocrit. After 20 min of HV in supine position, which lead to a decrease of the venous CO2 partial pressure by 19 Torr and to an increase of plasma epinephrine and norepinephrine levels, the plasma volume was reduced by 12.9%. The intravascular masses of total protein, albumin, and several other proteins decreased during HV but a similar decrease of these proteins was also observed during the control study (C), i.e., rest in supine position without HV. The differences between changes during HV and C were not significant, indicating that the loss of protein was not due to HV. It is concluded that acute HV leads to a rapidly reversible loss of a virtually protein-free solution from the vascular space. The red cell compartment participated in fluid shifts in that the mean red cell volume decreased by 2.2% (P less than 0.02 compared with C). Comparison with earlier work shows that addition of erythrocytes from the normal spleen does not play a part in the HV-induced increase of hemoglobin and hematocrit.     

Some haematological responses of normal and splenectomized rainbow trout Salmo gairdneri to a 12% blood loss

Anaemia can be induced in Rainbow trout by removing 12 % of the blood volume by cardiac puncture. Recovery from such an anaemia has been monitored in both normal and splenectomized fish. After the bleeding there is a decrease in red cell numbers, haematocrit and blood haemoglobin concentration. Although recovery in normal trout was not complete within 27 days, which was the period of the observations, it was seen as a replenishing of red cell numbers, haematocrit and blood haemoglobin concentration. Mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC), both depressed by the bleeding, did not appear to recover during the experimental period.
Splenectomy reduced erythropoietic intensity. After bleeding all the parameters studied were more depressed than those found in normal trout, and recovery was not noted. Blood haemoglobin and red cell numbers did not have the same post-bleeding pattern as normal individuals; MCH and MCHC values remained markedly depressed.
Splenic involvement was further demonstrated by bleeding trout for a second time from both normal and splenectomized groups. In contrast to the response of normal animals, there was a further deterioration of red cell numbers and blood haemoglobin concentration in splenectomized individuals.     

Distribution and metabolism of exogenous somatostatin in the rat

The mechanisms of clearance and degradation of injected [3H]somatostatin have been studied in the rat using octadecasilyl-silica extraction and HPLC separation methods.. Three apparent consecutive plasma half-lives of 1, 3 and 20 min were estimated following administration of a pharmacological dose. The initial rapid clearance was due to uptake by various tissue beds, mainly the large peripheral tissue masses muscle, skin and intestine which together accounted for 70% injected radioactivity at 1 min. By contrast the amounts taken up by liver and kidney were relatively small (less than 10%) despite the accumulation of higher concentrations. Massive degradation occurred following uptake and small fragments and amino acids were released into the circulation almost immediately. Inactivation by blood itself was negligible. A slow phase of decline observed at later times suggested a return of intact peptide from extravascular storage sites to sustain plasma concentrations.     

In vivo metabolism of brain natriuretic peptide in the rat involves endopeptidase 24.11 and angiotensin converting enzyme

The metabolism of brain natriuretic peptide (BNP) was studied in rats infused with 125I-BNP. During the infusion, the intact peptide was progressively converted to labelled degradative products, separated into nine peaks of radioactivity on HPLC, and accounting for approximately 70% of total plasma radioactivity at the plateau phase. After stopping the infusion, intact BNP disappeared with a half-life of 1.23 +/- 0.35 min whereas the labelled fragments accounted for progressively greater proportion of total activity. The degradation of BNP was significantly reduced by phosphoramidon (t1/2, 11.28 +/- 0.49 min) and captopril (t1/2, 6.99 +/- 0.34 min). A maximal effect was observed when both protease inhibitors were given simultaneously (t1/2, 15.3 +/- 0.48 min). When 125I-BNP was incubated in vitro with purified endopeptidase 24.11 (E-24.11) and angiotensin-converting enzyme (ACE), there was a time-dependent disappearance of the intact peptide associated with the generation of six labelled fragments, corresponding to fragments found in vivo. In serum the peptide was rapidly degraded with a half-life of 4.6 +/- 0.1 min, and the pattern of labelled fragments was similar to that observed during in vitro incubation with ACE. Captopril significantly reduced the rate of degradation of BNP in serum. The results allow to associate two define enzyme activities, namely E-24.11 and ACE, with the metabolism of BNP in vitro. They also indicate that, despite a close homology between ANP and BNP, the two peptides undergo different pathways of clearance.     

Blood volume determinations in sheep before and after splenectomy

Using 51Cr labelled RBCs, total blood volume, red cell volume and plasma volume were measured in fifteen adult, female, domestic sheep both before and after splenectomy. Eight of the fifteen animals studied were anemic. Statistical analyses revealed no significant differences in blood volume parameters whether animals were grouped together or separated into normal and anemic groups. We observed: (a) splenectomy produced modest reductions in blood volume parameters in 12 of 15 animals, (b) preoperative variability in blood volume parameters caused by release of sequestered RBCs from the spleen was eliminated after splenectomy, and (c) equilibration of 51Cr required at least 30 minutes in intact animals, but only 10 minutes in splenectomized animals. After volume parameters were normalized to body weight, they were found to agree closely with values reported previously. This study demonstrates the dynamic function of the sheep spleen in the regulation of blood volume.     

Fluid extravasation from spleen reduces blood volume in endotoxemia

We recently demonstrated that fluid is filtered out of the splenic circulation and into the lymphatic system. The current experiments were designed to investigate the importance of this route of fluid extravasation in endotoxemia. Lipopolysaccharide (LPS) was infused into conscious intact and splenectomized rats (150 microg x kg(-1). h(-1) i.v. for 18 h). In the intact rats, mean arterial pressure (MAP) fell from 101+/-2.4 to 88+/-3.9 mm Hg (n = 7) and then stabilized at about 90 mm Hg. Hematocrit rose from 41+/-0.9 to 45+/-0.4% at 40 min, at which time plasma volume had fallen from 4.7+/-0.12 to 4.0+/-0.05 ml/100 g body wt. In the splenectomized rats MAP did not fall and hematocrit did not rise. There also was no change in plasma volume, i.e., splenectomy prevented the hypotension and hemoconcentration customarily induced by LPS. In a second series of experiments, splenic arterial and venous blood flows were simultaneously measured in anesthetized rats infused with LPS (150 microg x kg(-1) x h(-1)). LPS increased splenic fluid efflux. We conclude that during endotoxemia the initial fall in circulating blood volume may be attributed to fluid extravasation from the splenic vasculature.     

Interdependence of bronchial circulation and clearance of 99mTc-DTPA from the airway surface.

The extent to which the systemic vasculature is involved in soluble-particle uptake in the conducting airways has not been studied extensively. In anesthetized, ventilated sheep, 6-10 microl of technetium-99m-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA) was delivered through a microspray nozzle to a fourth-generation airway. Perfusion of the cannulated bronchial artery was varied between control flow (0.6 ml x min(-1) x kg(-1)), high flow (1.8 ml x min(-1) x kg(-1)) or no flow (the infusion pump was stopped). Airway retention of the radioactive tracer was monitored using gamma camera imaging, and venous blood was sampled. During control perfusion, tracer retention at the site of deposition at 30 min averaged 20 +/- 6% (n = 7). With no flow, retention was significantly elevated to 32 +/- 8% (P = 0.03). In another group of sheep (n = 5) with a control retention of 13 +/- 4%, high flow resulted in an increase in tracer (25 +/- 4%; P = 0.04). Maximum blood uptake of tracer was calculated by estimating circulating blood volume and averaged 16% of total activity during control flow. Only during high-flow conditions was 99mTc-DTPA in the blood decreased (10%; P = 0.04). Most of the tracer was cleared by mucociliary clearance as visualized by imaging. This component was substantially decreased during no flow. The results demonstrate that both decreased and increased airway perfusion limit removal of soluble tracer applied to the conducting airways.     

High density lipoprotein metabolism in normolipidemic and cholesterol-fed rabbits.

New Zealand white rabbits were used to determine the compositional and metabolic changes induced in high density lipoproteins (HDL, rho = 1.063--1.21 g/ml) in response to cholesterol feeding. There was no change in total HDL cholesterol in plasma due to cholesterol feeding (12 weeks), but the triglyceride level was decreased to 29% of pretreatment values. Total protein content of HDL was decreased in response to cholesterol feeding, resulting in a significant increase in the cholesterol/protein ratio. There was a decrease in some isomer of the major apolipoproteins (A-I2) of HDL. The decay of radioactivity in HDL or its apolipoproteins was biphasic in both normolipidemic and hypercholesterolemic rabbits. The first phase was much more rapid than the second. The decay rates for the radioactivity in HDL depended upon the dietary status of the recipient animal.     

Effects of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on the cardiovascular system in sheep.

The cardiovascular effects of PACAP and VIP were studied in intact conscious sheep; PACAP (0.008, 0.04, 0.2, and 1.0 nmol/min) and VIP (0.07, 0.2, 0.6, and 1.8 nmol/min) were infused in conscious sheep for periods of 10 min. For each peptide there was a dose-dependent increase in heart rate. At the highest doses tested, pulse pressure and mean arterial pressure tended to increase and decrease, respectively. However, only the decrease in mean arterial pressure following the highest dose of VIP reached significance. At the highest doses tested, heart rate increased nearly threefold during the infusion while mean arterial pressure declined by 18.5%. In individual animals the decrease in blood pressure and increase in heart rate occurred simultaneously, so that we were unable to conclude whether the increase in heart rate was due to a baroreceptor reflex.     

Inosine metabolism in the rat

1. Uptake and subsequent metabolism of purine and ribose moieties was monitored after intravenous administration of doubly labelled inosine. 2. More than 95% was cleared from the plasma within 5 min, and 99% within 20 min. 3. Approx. 50% of the 160 mumol total was rapidly incorporated into liver and kidney. Kidney removed the greatest amount (21 mumol/g wet wt.), about 10-fold more than heart, lung or liver. Lung and heart accounted for only 3%. These tissues then lost radioactivity during the remainder of the experiment. Radioactivity in the skeletal muscle, in contrast, increased from 8% of the injected dose at 5 min to 40% at 60 min. 4. In liver, kidney, heart and lung there was a significant difference in the fate of inosine. After initial incorporation of inosine, kidney predominantly lost inosine; heart preferentially lost purines; lung preferentially lost ribose radioactivity; and in liver the ribose radioactivity was rapidly lost, whereas purine was retained. Some of the ribose moiety was metabolized to glucose, presumably in the liver, and then released into the blood. Ribose radioactivity (probably as glucose) and radioactive hypoxanthine accumulated in skeletal muscle throughout the experiment. 5. Inosine caused a rapid and prolonged increase in the blood glucose content, from 6 to 15 mM in 60 min. This was accompanied by a small increase in plasma insulin. 6. It is concluded that the purine and ribose radioactivity lost from the kidney, liver and other tissues becomes incorporated into skeletal muscle.     

Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium.

Slices of caruncular endometrium from steroid-treated ovariectomized sheep were incubated with myo-[2-3H]inositol to label tissue phosphatidylinositol. Effects of oxytocin were determined on the rate of incorporation of radioactivity into phosphatidylinositol and on the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol. Incorporation of radioactivity into phosphatidylinositol was linear during 2 h incubations; 10(-7) M (100 nM)-oxytocin caused a 2.8-fold increase in the rate of incorporation. In the presence of Li+, addition of 10(-7) M-oxytocin to slices in which phosphatidylinositol was pre-labelled caused mean increase of 40-fold in the incorporation of radioactivity into inositol mono-, bis- and tris-phosphates. Inositol 1,3,4-trisphosphate was quantitatively the major trisphosphate formed. The action of oxytocin on phosphoinositide hydrolysis was dose- and time-dependent, occurring at concentrations within the range observed in plasma during episodes of secretion in vivo, and with a time course comparable with that of the action of oxytocin on uterine prostaglandin production. The effect of oxytocin on incorporation of radioactivity into inositol phosphates was not affected by inhibitors of prostaglandin synthesis. Diacylglycerol 1- and 2-lipases in caruncular endometrium converted up to 72% of added 2-[3H]arachidonyldiacylglycerol into [3H]arachidonic acid during 30 min incubations at pH 7.0. Caruncular endometrium contained 1.49 mumol of phosphatidylinositol/g, representing approx. 0.2 mumol/g of phosphatidylinositol arachidonic acid. It is proposed that the stimulation of endometrial prostaglandin synthesis by oxytocin is accounted for by increased hydrolysis of phosphoinositides to diacylglycerol and inositol phosphates with subsequent release of arachidonic acid from diacylglycerol.     

Respective roles of kallikrein and endopeptidase 24.11 in the metabolic pathway of atrial natriuretic peptide in the rat.

The metabolism of atrial natriuretic peptide (ANP) and Cys-105-Phe-106-cleaved ANP (ANP) was studied during constant infusion of 125I-labelled peptides in rats. Analysis of circulating radioactivity indicated rapid clearance of ANP and ANP', with mean half-lives of 0.42 and 1.04 min respectively. H.p.l.c. fractionation of plasma taken during the infusion of labelled ANP revealed the presence of three radioactive fragments, the major one co-eluting with 125I-ANP'. These fragments correspond to cleavage products previously found to be generated in vitro by the action of endopeptidase 24.11 (E-24.11). On evaluating the effects of peptidase inhibitors, a significant increase in the half-life of ANP was observed with phosphoramidon (t1/2 7.8 min) and aprotinin (t1/2 5.4 min). A maximal inhibition of ANP degradation was obtained when both inhibitors were given simultaneously (t1/2 15 min). In blood samples taken during infusion of 125I-ANP and phosphoramidon, the intact peptide accounted for more than 90% of total circulating radioactivity, and no cleavage product was present in detectable amounts. Phosphoramidon had no effect on the metabolism of infused ANP'. In contrast, when 125I-ANP' was infused together with aprotinin, the rate of degradation of the infused peptide was reduced by more than 80%. It is proposed that two different peptidase activities, E-24.11 and a kallikrein-like proteinase, are responsible for the cleavage of ANP in the circulation. The Cys-Phe-cleaved ANP would in turn be degraded by kallikrein and not by E-24.11.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.