The effect of acute changes in blood flow on local lymph flow in the limbs of anesthetized sheep.

A study was made of the effects of acute changes in local blood flow on lymph flow in the feet of anesthetized sheep. Lymph outflow pressure and flow and venous pressure were measured in cannulated vessels draining the foot region. Local volume changes were also measured. Acute reductions in blood flow were produced by sudden occlusion of the circulation to the limbs with a pneumatic cuff for periods of up to 1.5 hours. During the first 10 min of occlusion, there was little change reaching about 25% of its control value after 40 min of arrest. On release of the circulation, lymph flow rose almost immediately to levels above the control value, resulting in a hyperlymphia whose size and duration was related to the duration of the circulatory arrest and the limb volume changes that followed. The results indicated that lymph flow can continue, albeit at reduced rates for long periods after circulatory arrest and that during reactive hyperaemia, there is a brisk hyperlymphia whose size and time course is similar to that of the limb volume changes.

Lymph flow in sheep limbs during local exposure to subatmospheric pressure.

1. Lymph flow and pressure were measured via cannulae inserted into afferent lymphatics draining the feet of anaesthetized sheep. 2. When the cannula outlet was at limb level, local exposure of the limb to graded decreases in ambient pressure caused graded increases in lymph flow with pressure values down to -50 mmHg. 3. When the cannula outlet was lowered below limb level to offset the negative pressure gradient imposed on the lymphatic vessels by suction, lymph flow rose progressively with decreasing ambient pressure values down to -70 mmHg. 4. When negative pressure gradients were imposed on the lymphatic vessels by raising the lymphatic cannula outlet in progressive steps above limb level, the vessels were able to expel lymph against gradients of up to 50 mmHg but lymph flow was greatly reduced against a gradient of 70 mmHg. 5. The results suggest that subatmospheric pressure may affect local lymph flow in two ways. By increasing blood capillary transmural pressure it may increase lymph flow by increasing tissue fluid formation. By imposing a negative pressure gradient along the lymphatics it may decrease lymph flow, especially at the most negative pressures, and the lymph flow response to subatmospheric pressure may be the algebraic sum of both effects.

Comparison of fluid transport systems in lymphatics and veins.

In the anesthetized sheep, pressure pulses generated in the feet are transmitted downstream in the veins but not in the lymphatics at normal intralymphatic pressure. When the sheep is tilted on a tilt table, gravitational changes occur in venous pressure but not in the pressure in adjacent lymphatics. These results suggest that in limb lymphatics, unlike limb veins, the column of fluid is incomplete. This makes extrinsic pumping less effective for propelling fluid in lymphatics than in veins. At normal intralymphatic pressures, intrinsic pumping seems to be mainly responsible for lymph propulsion. The incompleteness of the fluid column in lymphatics might also protect these vessels against the hydrostatic problems experienced by veins during gravitational stress.

Resistance in the sheep's lymphatic system.

Saline was infused in a downstream direction into the afferent lymphatics in the metacarpal region of anesthetized sheep. The changes in inflow pressure were measured over 10 min periods with flow rates ranging from 10-2000 microliters/min. Flow rates in the physiological range generated mean pressures of about 30 mmHg and flows of 1 ml/min generated mean pressures of about 60 mmHg. Resistance was relatively high at flow rates in the range of 10-15 microliters/min but sharply decreased above that and was relatively constant at flows greater than 500 microliters/min. Adding isoprenaline (1 microgram/min) to the infusate reduced spontaneous contractile behavior in the infused system and lowered the resistance at the lower flow rates. It is concluded that the peripheral lymphatic system in the sheep offers substantial resistance to lymph flow and that substantial intralymphatic pressure is needed to return lymph from the periphery especially at higher flow rates.

Interference in the cytochemical assay of plasma vasopressin by a circulating factor induced by salt-loading in man.

Infusion of hypertonic saline into six normal volunteers caused an increase in plasma osmolality from 286.8 +/- 1.7 (mean +/- S.E.M.) to 307.6 +/- 2.6 mosmol/kg (P less than 0.001), a 7.1% increase in estimated blood volume, a rise in plasma immunoreactive arginine vasopressin (AVP) concentrations from 1.3 +/- 0.2 to 12.7 +/- 3.6 pmol/l (P less than 0.001) but no change in plasma AVP concentrations (2.1 +/- 0.9 and 1.9 +/- 1.3 pmol/l) as measured by a cytochemical technique based on the ability of AVP to stimulate rat renal medullary Na+/K+-ATPase activity. Addition of synthetic AVP to plasma obtained before, during and after hypertonic saline infusion also failed to stimulate Na+/K+-ATPase activity. The results suggest that infusion of hypertonic saline interfered with the cytochemical assay for AVP by inhibiting AVP-stimulated medullary Na+/K+-ATPase activity. We conclude that the use of this cytochemical method to detect plasma AVP has severe limitations under these experimental conditions.

Development of a cytochemical assay for plasma vasopressin: application to studies on water loading normal man.

A cytochemical assay has been developed to measure human plasma arginine vasopressin. It is based on the stimulation of Na+-K+, ATPase activity located in the outer medulla of the rat kidney, and is capable of detecting very low plasma arginine vasopressin concentrations, limit of detection 0.01 pmol/l. Specificity for vasopressin stimulation of the enzyme is conferred on the assay by the use of specific vasopressin antiserum. Index of precision of the assay is 0.21. Degradation of arginine vasopressin in plasma in inhibited by phenanthroline. Samples may be stored up to 8 weeks at -70 degrees C. Intra- and inter-assay coefficients of variation were 22% (n = 8) and 104% (n = 12), respectively. A sustained water load in eight healthy male adults caused a fall in plasma osmolality from a basal of 286.5 +/- 2.0 (mean +/- SEM) to 279.2 +/- 2.4 mmol/kg after the load (P less than 0.001), which was associated with a reduction in urine osmolality from 867 +/- 54 to 69 +/- 3 mmol/kg. Plasma immunoreactive arginine vasopressin fell from 1.3 +/- 0.3 pmol/l to become undetectable (less than 0.3 pmol/l), but plasma cytochemical arginine vasopressin decreased from 0.96 +/- 0.14 to 0.07 +/- 0.02 pmol/l. There was a curvilinear relationship between plasma osmolality and plasma cytochemical arginine vasopressin, which militated against the concept of an osmotic threshold for vasopressin release.

Rat hypothalamic extract inhibits vasopressin-stimulated Na+-K+-ATPase activity in the rat renal medulla.

Arginine vasopressin stimulates Na+-K+-ATPase activity located in the rat thick ascending limb of Henle's loop. mammalian hypothalamus appears to produce a factor capable of inhibiting Na+-K+-ATPase activity in a variety of tissues. The effect of a purified rat hypothalamic extract with and without AVP on rat renal Na+-K+-ATPase activity was evaluated by a cytochemical technique. The hypothalamic extract alone failed to affect basal Na+-K+-ATPase activity throughout renal segments after 10 min exposure. Na+-K+-ATPase activity stimulated by AVP (1-10 fmol l-1) for 10 min was inhibited by rat hypothalamic extract over the concentration range 10(-7)-10(-3) U ml-1 in a dose-dependent manner. Complete inhibition of AVP-stimulated Na+-K+-ATPase activity occurred at a hypothalamic extract concentration of 10(-3) U ml-1. Only Na+-K+-ATPase activity located in the renal medullary thick ascending limb was influenced by the rat hypothalamic extract.

Prolactin stimulates Na+-K+-ATPase activity located in the outer renal medulla of the rat.

The effect of rat prolactin on rat renal Na+-K+-ATPase activity was investigated by a cytochemical technique. Rat prolactin caused stimulation of Na+-K+-ATPase activity only in the outer medulla of the kidney, and not in renal cortical structures. Peak enzyme activity in cultured rat renal segments occurred after tissue had been exposed to rat prolactin for 2 min, and the time of maximal stimulation did not vary with the concentration of prolactin. There was a curvilinear response in Na+-K+-ATPase activity over the rat prolactin concentration range, 0.04-40 ng/l, but higher prolactin concentrations caused inhibition of enzyme activity. Na+-K+-ATPase response was totally blocked by specific rat prolactin antiserum. Human prolactin had no consistent effect on rat medullary Na+-K+-ATPase activity. Addition of specific tri-iodothyronine and arginine vasopressin antisera to rat prolactin was without effect, confirming that the stimulatory action of rat prolactin on Na+-K+-ATPase was not due to contamination with these hormones which are known to stimulate this enzyme.

The stimulation of Na+, K+-ATPase activity in the medulla of the rat kidney by [arginine] vasopressin and its analogues.

Total and Na+, K+-ATPase activities have been measured in sections (10 micron thick) from blocks of rat kidney cultured for 5 h at 37 degrees C, pH 7.5, in Glasgow Eagle's Minimum Essential Medium. Synthetic [arginine]vasopressin [( Arg]VP) stimulated ATPase activity in the thick tubular cells of the renal outer medulla over the concentration range 0.01-10 fmol/l, but failed to affect ATPase activity in the proximal and distal convoluted tubules of the cortex. The increase in medullary total ATPase activity induced by [Arg]VP and its analogues was wholly due to stimulation of Na+, K+-ATPase activity. Stimulation of medullary ATPase activity was blocked by [Arg]VP antiserum. The [Arg]VP analogues desmopressin, [lysine]vasopressin, [arginine]vasotocin and oxytocin stimulated medullary Na+, K+-ATPase activity, the three last-named analogues being considerably less potent than [Arg]VP.

Stimulation of rat renal Na+, K+-ATPase activity by thyroid hormones.

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+,K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+,K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+,K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 -1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.