Abstracts of papers presented at the twenty-fourth annual meeting of the microcirculatory society. April 11–12, 1976. Anaheim, California

MICROVASCULAR RESEARCH II, 115-131 (1976)

Abstracts Annual

of Papers Presented at the Twenty-Fourth Meeting of the Microcirculatory Society April I I-12, 1976 Anaheim, California

1. Quantification of the Responseof the Microvasculature in Experimental Mammary Tumors in C3H Mice to Vasoactive Drugs. W. P. BARRETT,H. W. PUFFER,J. C. KOFOED, AND N. E. WARNER, Department of Pathology, University of Southern California, Los Angeles, California 90033. We have developed a vasoconstriction index whereby the relative activities of various vasoactive drugs may be compared. Using epi-illumination apparatus, the microvasculature of the experimental mammary tumors in C3H mice was observed and measured using a Leitz trinocular microscope equipped with a Leitz filar micrometer. The diameter of the most prominent venule and arteriole was measured prior to the topical application of epinephrine hydrochloride, isoproterenol hydrochloride, levarterenol bitartrate, propranolol hydrochloride, and phenoxybenzamine hydrochloride. The vasoconstriction index (VI) was calculated using the formula VI = VD*/VD,, where VDI is the diameter of the vessel prior to application of the drug and VD2 is the diameter of the vessel after application of the drug. The vasoconstriction indexes, for example, a vasoconstriction index of 0.97 for venules and 0.24 for arterioles in small (less than 0.5 cm diam) tumors in mice and an index of 0.97 for venules and 0.95 for arterioles in large (greater than 1 cm diam) tumors using epinephrine hydrochloride, 1: 100,000, will be reported for the above drugs in C3H mice. 2. Extravascular Plasma Albumin Pool in Kidney. D. R. BELL, J. E. STORK,AND G. G. PINTER,Department of Physiology, University of Maryland, School of Medicine, Baltimore, Maryland 21201. The extravascular albumin pool in rat renal cortex was estimated by measuring the distribution volumes of two equivalent and distinguishable tracers of plasma albumin. One of these, tagged with I-125, was permitted to equilibrate over a period of 2 hr, and its distribution was taken as indicative of the total albumin pool. The other tracer, labelled with 1-131, was allowed to circulate for 3-1 min, sufficient to ensure a homogeneous mixing with the circulating blood plasma. The volume of distribution of the second tracer was larger than the intravascular volume as evidenced by an increasing activity in renal lymph. That amount of the second tracer which crossed into the interstitium during this short interval was estimated from a kinetic model of the interstitial albumin pool by using the measured values of the mean transit time of albumin molecules from plasma to renal lymph and the specific activity vs time relationships of albumin in plasma and lymph. These data were obtained from separate studies under identical experimental conditions. The extravascular distribution volume was calculated as the corrected difference between the steady-state and time-dependent tracer distribution volumes. A value of 1.71 k 0.22 @EM, N = 23 rats) ml/100 g of renal cortex was obtained, which corresponded to 13.6% of the total cortical albumin pool. 3. Venous-Arteriolar Response in Striated Cremaster Muscle in the Rat. DAISY BENITEZ,AND SILVIO BAEZ, Instituto Nal. de Cardiologia, Mexico; and Albert Einstein College of Medicine, Bronx, New York, 10461. A previous report (Blood Vessels 11, 260, 1974) has described the response of arterioles (Artl) to systemic venous pressure elevation (Vp el) in rat mesentery. The aim of this work was (a) to determine whether morphologically similar microvessels of striated muscle (Cr ms) would respond to Vp el in Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

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the same manner; and (b) to explore the nature of the response. Under nembutal anesthesia (30 mg/kg, im) the Cr ms was prepared and mounted for microscopy. The ipsilateral femoral artery and vein were cannulated, and the tip of the cannulas was advanced near the origin of cremasteric artery and vein. Except for the arterial supply and venous drainage, the tissue was isolated from all other vascular connections. Systemic B.P. and Vp were recorded, Vp el was done by partial occlusion of the external iliac vein, and changes in arteriolar diameter (i.d.) were measured by image-splitting. In 7 out of 11 rats Vp el (4.7 f 1.9 mm Hg) resulted in no change in systemic B.P., and in 3 animals in minimal (2.5 mm Hg) B.P. decline. In the remaining four rats, Vp el (5.1 f 2.7) was accompanied by B.P. decrease (9.4 & 4.8 mm Hg). In all experiments Vp el resulted in variable arteriolar constriction and degrees of capillary blood flow curtailment. Diameter during Vp el was measured in 29 arterioles (43.3-7.0 pm id.). In the seven rats with minimal or no change in B.P., the arteriolar constrictions (%) were: Artlr (n 6), 22.4; Art& (n 5), 34.4; and metarteriole (n 7), 21.3. The arteriolar diameter and capillary blood flow changes returned toward control upon release of the stress of Vp el. Dibenzyline (0.1&0.25 mg/ml) abated the response. The results show that arteriolar constriction to Vp el present in striated muscle depends in part on an intact a-receptor force. (Work supported by NIH Grant No. HL-06736.) 4. Flow Improvers Prevention of Microcirculation

Changes Induced by Ionizing Radiation

to the Liver.

H. I. BICHER, AND N. KAUFMAN, Departments of Pharmacology and Radiology, University of Arkansas Medical School, Little Rock, Arkansas 72201. Platelet aggregation and adhesiveness, as well as TpOz responses to hypoxia and hyperoxia, were measured as microcirculation parameters in beagle dogs subject to Co60 ionizing radiation to a dose of 4600 rad in 5 weeks. Simultaneously, changes in blood chemistry, liver scans, and microscopy were also determined. Marked changes in the studied parameters in the postradiation period lead to the conclusion that radiation liver damage is at least in part mediated through microcirculation disturbances. As a part of these studies, the normal regulatory responses of liver TpOl to respiratory upsets in O2 supply were determined. The existence of an “autoregulatory” mechanism, similar but less accurate than that described for brain (Bicher et al., Amer. J. Physiol. 224,275,1973 ; Bicher, Microvasc. Res. 8, 291, 1974) was determined. The reoxygenation time (RT) (period of time required for TpOz to return to the original level after a short period of anoxic anoxia) was found to be an adequate parameter by which to determine the ability of the microcirculation to deliver OZ to tissue. During 0, breathing, TpOz rises to about double its original value (as described by Cater and Silver, Acta Radiol. 53, 233-256, 1959). Infusions of low molecular weight dextran (Rheomacrodex) during the radiation period prevented the microcirculation changes mentioned above. Determination of Aggregation Force in Red Cell Rouleaux. SHU CHIEN, AMV LAN-PING SUNG,SYNGCUKKIM, ALLAN BURKE,AND SHUNICHI USAMI, Department of Physiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.

5. Microrheological

The molecular bridging force causing aggregation of red blood cells (RBC) was estimated from the shearing force required to disaggregate rouleaux under microscopic observation. Normal human RBC were suspended in Ringer solutions containing various concentrations and molecular weights of dextrans (Dx). A flow channel (modified from Hochmuth et al., Biophys. J. 13, 747, 1973) was filled with the cell suspension. The flow channel was connected to a pump with a continuously variable speed for the infusion of the same Dx solution as that used to suspend the cells. The pressure drop across the flow channel at various infusion rates was monitored. The shear stress acting on the rouleaux was calculated from the pressure drop and the channel geometry, and correlated with the degree of rouleaux disaggregation determined from microscopic pictures. The stress-disaggregation curves are sigmoidal. The shear stress required for 50% surface separation (r,J varied directly with Dx molecular size and showed a biphasic relation with Dx concentration (maximum 51/zat 46 g%). This study, in which the geometric relation between shear flow and RBC rouleaux is well defined, yields quantitative information on force balance in RBC aggregation. (Supported by Grants Nos. HL 07114 and HL 16851 from NHLI, and U.S. Army Contract No. DADA 17-72-C-2115.)

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of Single Mesenteric Capillaries. C. CRONE,AND J. J. FRIEDMAN,Institute of Medical Physiology, Dept. A., University of Copenhagen, Copenhagen, Denmark; and Department of Physiology, Indiana University, Indianapolis, Indiana 46202.

6. Potassium Permeability

Organ studies ofcapillary permeability are plagued by the numerous assumptions required to conduct an estimate. Many of these assumptions can be obviated by adopting a single capillary experimental model. The potassium permeability was determined in single capillaries of the frog mesentery by means of a microadaptation of the indicator diffusion method. The frog mesentery is exposed, transilluminated, and irrigated at room temperature with frog Ringer-albumin solution. Under microscopic manipulation, a 5-10 PM injection micropipette and l-2 pM upstream and downstream K-sensitive ion-exchange microelectrodes are introduced into the capillary flow stream. A microbolus of 50 mM KC1 is injected, and the upstream and downstream electrodes, respectively, record the time-concentration input and output from the capillary segment between them. The initial extraction of potassium, E, is defined as the relative differences between the areas under the early part of the two curves. Interelectrode surface area is calculated from the capillary radius (r) and length (L) dimensions. Capillary flow, Q, is calculated as the interelectrode capillary volume (ar2 .L) divided by the difference in appearance times (tl - tl) of the potassium bolus at the two electrodes, assuming plug flow. Permeability, P, is then calculated as P = -(Q/.S)ln(l - E). For the first 11 successful determinations P averaged 1.6 x 10e3cm set-‘. This value is higher than figures reported from organ studies. The basis for this discrepancy will be discussed. I.

Effective Osmotic Pressure of Plasma Proteins Across Large Cylindrical Pores. J. DHAR, H. H. LIPOWSKY,AND H. J. GRANGER,Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi; and AMES-Bioengineering, University of California, San Diego, La Jolla, California 92093.

The purpose of this study was twofold: (1) to ascertain the magnitude of the effective osmotic force generated by physiological concentration gradients of plasma proteins across large pores of uniform geometry; and (2) to compare the experimental results with the theoretical predictions of the equivalent pore model of membrane permeability. The reflection coefficients of bovine serum albumin and gamma-globulin with respect to polycarbonate membranes (Nuclepore) containing uniform cylindrical pores 250 A in radius were determined by two methods. The first consisted of comparing the volume flow induced by an oncotic pressure gradient with the transmembrane flow generated by a hydraulic pressure differential of the same magnitude. The second approach involved comparison of the osmotic pressure obtained with the polycarbonate membranes mounted on an osmometer with the oncotic force measured with membranes impermeable to plasma proteins. The reflection coefficients for albumin and gamma-globulin averaged 0.03 and 0.08, respectively. These results suggest that (1) the effective oncotic pressures of plasma proteins across large pores are negligible in the physiological concentration range, and (2) the equivalent pore model over-estimates the reflection coefficient of plasma proteins in large pore systems. (Supported by Grant-in-Aid 72-947 from the American Heart Association and USPHS Grant No. HL-10881.) 8. Blood Flow Velocity during Rest and Postocclusive Reactive Hyperemia in Human Skin Capillaries. B. FAGRELL, A. FRONEK, AND M. INTAGLIETTA, AMES-Bioengineering, University of California,

San Diego, La Jolla, California 92093. Red blood cell velocity in human nail fold capillaries during rest and reactive hyperemia was studied by television microscopy. The method measures the time delay between signals which originate from erythrocytes and plasma gaps passing two video photometric windows, positioned along a capillary. The data were analyzed by manual evaluation of the time lag between the densitometer signals and by measuring their delay by cross-correlation technique. The average resting flow velocity in the examined subjects was 0.5 mm/set (SD ? 0.2). In most subjects spontaneous fluctuations of the velocity were observed at a frequency of approximately 6/min. By means of a miniaturecuff at the base of the investigated finger, a I-min stop of the arterial blood supply to the skin area studied was performed. An average peak increase of 250% and a 50% decay time of 15 set were recorded during the reactive

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hyperemia. It is suggested that these indices may be useful for studying changes of the skin microcirculation in patients with different vascular disorders. (Supported by Grants Nos. HL 12493 and TW 00249 from USPHS.) 9. Effects of Transcapillary Fluid Movement on Solute Exchange in Isolated Dog Hindlimb. BERNARD P. FLEMING,AND JOHNN. DIANA, Department of Physiology, and Cardiovascular Center, University of Iowa, College of Medicine, Iowa City, Iowa 52242. The existence of simultaneous transcapillary movements of fluid and solute via identical pathways through the endothelium raises the possibility that coupling between this volume flow and solute fluxes may occur. To study this phenomena in isolated dog hindlimbs, we employed the isogravimetric and single-injection multiple indicator dilution techniques. Venous indicator dilution curves were generated in the isogravimetric condition and at various rates of filtration and absorption. In hindlimbs vasodilated with papaverine, net filtration or absorption had little or no effect on maximal extractions of urea, sucrose or polyethylene glycol(5000 MW). These experimental findings agree with the predictions of a Renkin-Crone model of transcapillary solute exchange which was modified to contain a “solvent drag” term. The expression derived for the extraction of a test solute is given by E = 1 - exp [-&S/F) - (1 - a)(&S/2F)I, where PS is the permeability-surface area product for the test solute, D is the reflection coefficient, F is the appropriate flow, and JrS is the net rate of fluid movement. For example, using approximate experimental values for urea, the ratio of the second term in the brackets to the first term is 0.04. Thus, “solvent drag” effects on extraction determinations seem to be minimal. (Supported by Grants Nos. HL 16697 and HL 14388 from NHLI.) 10. Effect of Dose Level of Histamine on Mesenteric Microvascular Permeability. JAMESR. Fox, AND HAROLDWAYLAND,Division of Engineering and Applied Science, California Institute ofTechnology, Pasadena, California 91125. Bovine or rat serum albumin tagged with fluorescein isothiocyanate was injected iv (25 mg/kg) in cat or rat while the mesentery was viewed under a television fluorescence microscope. In rat, topically applied histamine ranging in concentration from lo-100 fig/ml of tris-buffered physiological solution always produced tracer efflux from veins and venules within l-2 min. A concentration of 5 pg/ml produced either no observable response, or tracer efflux at only a few loci along the veins or venules. Concentrations of I or 2 pg/,ul never produced an observable tracer efflux. When multiple doses of histamine were used, starting at 1 or 2 pg/ml and increased in steps, no response was observed even when doses exceed 10 pg/ml. In cat mesentery, initial histamine doses of 1,5, 10, and 100 fig/ml always produced tracer efflux from veins and venules, while 0.1 pg/ml produced efflux from only a few loci along the veins or venules. Thus the histamine concentration required to produce observable permeability changes for albumin in the microvasculature is about 5 pg/ml in the rat and 0.1-0.5 pg/ml in the cat, but these values are considerably higher if the tissue has previously been exposed to histamine below the threshold for efflux. (Supported by USPHS Grant No. HL 08977 and a grant from the John A. Hartford Foundation.) 11. Scanning Electron Microscopy of Superfused Mesentery. WALLACE G. FRASHER,KENNETH OSHIMA,AND CAROLUNRUH, University of Southern California School of Medicine, Los Angeles, California 90033. Peine and Low (J. Anat. 142, 137-158,1975) reported striking changes in surface structure by SEM of cardiac endothelium after brief exposure to Ringers Solution. We have studied cat mesentery following in vivo immediate fixation with glutaraldehyde and with fixation after varying times of exposure to superfusing Ringer-Tyrodes solution. Specimens were critical point dried with CO2 from aIcoho1. The control mesentery surface is smooth and without prominent cytoplasmic relief structures, Nuclei are oval regularly oriented and of low relief. Cell boundaries cannot be defined from the surface. In contrast saline-exposed tissues show profuse microvilli particularly supra-nuclear. Nuclei appear to condense and bulge prominently. Cell boundaries can be discerned by ridging and

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occasional separation. After prolonged exposure in a flow-field smooth bare areas occur which may represent exposed basement membrane. These specimens are to be studied by TEM to determine morphology of cell junctions. It is suggested that these changes probably imply an altered diffusiona? barrier at the mesothelial surface in superfused tissue. (Supported in part by NIH Grant No. HL 11153.) Arterioles to ExtrauascularpH Changes. J. H. GREENRERG, M. REIVICH, AND A. NOORDERGRAAF, Department of Neurology and Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19174.

12. Response Time ofcerebraf

In the course of the development of a mathematical model for cerebral blood flow control in hypercapnia, it became necessary to examine the speed with which cerebral arterioles could alter their diameter in response to a change in the pH of the fluid in which they were bathed. In 14 anesthetized cats a section of the parietal cortex was exposed by trephination, the dura was carefully removed, and the brain was bathed in mineral oil. Glass micropipettes (IO-15 pm) were sharpened and filled with mock cerebrospinal fluid (CSF) of the desired pH. The tip of the micropipette was positioned in the perivascular space of the pial arteriole (25-100 pm) with a micromanipulator, and the solution was infused at a constant rate (0.85 &min) during which time the field containing the vessel of interest was photographed repeatedly through an operating microscope. The temporal behavior of the vessel diameter changes was obtained from measurements made on the resulting negatives. An antimony micro pH electrode was placed in the vicinity of the vessel, and the micropipette to monitor the time course of the pH change was imposed on the vascular wall. Vascular responses were obtained using solutions with a wide range of pH. These studies indicate that cerebral arterioles in vivo dilate with a time constant of less than 15 set to a change in the pH of the solution in which they are bathed. This upper limit of response time was shown to be independent of vessel size. (Supported by NIH Prog. Project Grant No. NS10939-02), NIH Grant No. HL 10,330,and Medical Research Council of Canada Studentship.) Receptors in the Cat Mesenteric Circulation. P. H. GUTH, AND E. SMITH, Wadsworth VA Hospital, and University of California, Los Angeles, Los Angeles California 90073.

13. HI and Hz Histamine

Variations in distribution and function of histamine receptors have been described in different portions of the vascular system in different species. The aim of this study was to determine the types and functions of histamine receptors in the cat mesenteric circulation. Mesenteric flow was measured via an electromagnetic probe on the superior mesenteric artery and arterial pressure via a cannula in the carotid artery in 11 anesthetized cats. Histamine (H), mepyramine (M) (an Hi antagonist), and metiamide) (MET) (an H2 antagonist), were infused alone and in combinations into the superior mesenteric artery. Doses employed (micrograms per minute) were: H, 0.25-1.0; M, 0.25-1.0; and Met 3-12. In two cats submucosal (SM) arteriolar diameter changes also were measured using in uivo microscopy with an image-splitting technic. M or Met alone or together had no effect. H produced a dose-related decrease in mesenteric vascular resistance. M produced a 23 f 10% inhibition, and M + Met a 49 + 26 % inhibition of the H effect. Met alone had no effect on the H response. The preliminary in uivo microscopy studies revealed SM arteriolar diameter increase and mesenteric vascular resistance decrease with H. Both responses were inhibited by M and were further inhibited by M + Met, but not by MET alone. Conclusion: H, and H, histamine receptors, both mediating vasodilatation, are present in the cat mesenteric circulation. Failure to demonstrate an H, effect in the absence of Hr blockade suggests predominance of H, receptors. Similar conclusions have been drawn by others from studies of the whole circulation. Endothelial Reactions to Hypertension. D. W. HAACK, P. T. JOKELAINEN, J. A. G. RHODIN, AND J. G. SIMPSON,Department of Anatomy, University of Michigan Medical School, Ann Arbor, Michigan 48109.

14. Early Arteriolar

Eyes obtained from young pigs, which had been hypertensive for 4-6 weeks after unilateral nephrectomy and DOCA implantation, and from control animals were prepared for electron microscopy by routine methods. Examinations revealed an apparent increase in the permeation of inter-endothelial

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junctions by tracers, interruption of the junctions by focal dilations, and the appearance of large vacuoles in the endothelial cells of arterioles from hypertensive animals. This increase in the permeability of the blood-retinal barrier may be further expressed in the swelling of the intercellular spaces and separations of the external laminae in the vessel walls. Myo-endothelial junctions remain intact in spite of the distention of the extracellular spaces. Although chronic hypertension produces changes in the tunica media, the early effects of hypertension are on the endothelium. The impenetrability of the blood-retinal barrier in control arterioles and the presence of numerous myo-endothelial contacts in both control and hypertensive vessels may indicate that the effects of pressure and circulating vasoactive agents on smooth muscle cells may be mediated by the endothelial cells. (Supported by Grants Nos. HL 18575-01and 1 F32 HL05070-01.) in CollectingLymph Vessels. A. R. HARGENS,ANDB. W. ZWEIFACH,AMES Bioengineering, University of California, San Diego, La Jolla, California 92093.

15. Myogenic Contractility

Contracting lymph vesselsin mesentery which collect fluid from the intestine were used to investigate whether contractility is myogenic or of other origin. Lymphatic diameter and micropressures were continuously recorded in 18 Sprague-Dawley rats and 5 guinea pigs under normal conditions and during micromanipulation of lymph pressure. The effects of various drugs and protein solutions on the contractile mechanism were also studied. Spontaneous activity involves characteristic changes in intralymphatic pressure together with opening and closure of upstream and downstream valves. Contractions are often irregular but are predictable by noting the elevation of micropressure to the threshold pressure level. Mechanical obstruction upstream of the lymph vessel reduces contraction frequency and lymph pressure amplitude. Downstream occlusion increases contraction frequency and micropressure. Microinjection or withdrawal of fluid causes contractility to rise and fall, respectively. Contraction rate was not affected by the level of anesthesia. Suffusion of the preparation with a solution containing IO pg of norepinephrine, isuprel, or 10% serum albumin stops spontaneous contractions. A 10 bugsuffusion of papaverine has no effect. The data suggest that the contractile mechanism for these vessels responds primarily to pressure fluctuations in the particular lymphatic unit. (Supported by Grant No. HL-10081 from NHLI.) 16. Effects of Intracranial Hypertension and Brain Distortion on Blood-Brain Barrier. A. F. HECK, M. BROCK,M. HASUO,M. FURUSE,ANDH. DIET& University of Maryland, Department of Neurology, Baltimore, Maryland 21201; and Neurochirurgische Klinik, Medizinische Hochschule, Hannover, BRD. The blood-brain barrier to endogenous serum albumin labeled with Evans blue was studied by standard fluorescence microscopic techniques in cats subjected to intracranial hypertension and brain distortion produced by epidural balloon expansion. Intracranial pressure, blood pressure, blood gases, and pH were monitored. Systematic evaluation revealed numerous areas of extravasation of Evans blue-albumin reaction product and stained microvessels, sometimes associated with congestion or hemorrhages, in the cerebrum, brain stem, and upper spinal cord, which were not found in anesthetized and sham-operated control animals. As in previously reported studies, lesions were mainly remote from the site of increased pressure induction. Of particular interest was the selective involvement, unilaterally or bilaterally, of microvessels in the supraoptic and paraventricular nuclei of the hypothalamus in most experimental animals, this relating perhaps to the inappropriate antidiuretic hormone syndrome not infrequently encountered following head injury and/or intracranial hypertension. (Supported by Grant No. NS 06779, NJNCDS, and by the Alexander von Humboldt Stiftung, BRD.) 17. Viscosity of Human Red Cell Membrane in Plastic Flow. R. M. HOCHMUTH(Sponsor, S. P. SUTERA),

Department of Chemical Engineering, Washington University, St. Louis, Missouri 63130. The rate of growth of membrane filaments (“tethers”) permanently (plastically) pulled from surfaceattached red cells by the action of fluid shear forces has been measured in a variety of experiments using a parallel plate flow channel. In addition, this plastic growth phenomenon has been analyzed

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recently (Evans and Hochmuth, Membrane viscoplastic flow. Biophys. J. 16, 1976), based on the assumption that the membrane behaves as a two-dimensional Bingham material (a material with a rate of deformation which is proportional to the magnitude of the difference between the membrane shear and the yield shear). The combined experimental and theoretical results give values of lo-* dyn/cm for the yield shear and lo-* dyn set/cm @oise.centimeter or surface poise) for the “plastic” viscosity. Division of the plastic viscosity value by 10m6cm yields a “pseudo” three-dimensional value of IO4 poise. Published values for membrane lipid viscosity are l-10 poise. Thus, since the value for the plastic viscosity greatly exceeds thevalue for membrane lipid viscosity, it is concluded that membrane plastic flow characterizes the plastic deformation of structural membrane proteins in situ. (Supported by Grants Nos. HL 12839 and 70612 from the NHLI.) 18. Mechanism of Heparin-Induced

Red Cell Aggregation. KUNG-MING JAN, AND SHU CHIEN, Department of Physiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.

Heparin, an acid mucopolysaccharide commonly used as an anticoagulant, can cause aggregation of platelets and red blood cells (RBC). This study was designed to investigate the mechanism of heparininduced RBC aggregation. RBC aggregation in heparin-saline solutions was quantified by microscopic observation. A rise in concentration of commercial heparin (Sigma Co.) above 1 g% caused a progressive increase in RBC aggregation. The effectiveness of purified beef lung heparin fractions (kindly supplied by Dr. J. A. Cifonelli) in causing RBC aggregation increases with their molecular weights (6 x 103-15.5 x 103). Lowering the ionic strength of the heparin solution, which reduces cationic screening at RBC surface and increases the molecular length of heparin, enhanced RBC aggregation. Removal of sialic acid from RBC surface by neuraminidase markedly reduced the heparin-induced aggregation. Furthermore, studies on electrophoretic mobility of RBC showed a progressive increase of zeta potential with rising heparin concentration. These results suggest that the binding of heparin to sialic acids on adjacent RBC surfaces forms molecular bridges for RBC aggregation. These studies not only serve to elucidate the basic mechanism of cell-cell interaction mediated by macromolecules, but also to provide insights into the molecular structure of heparin. (Supported by NHLI Grant No. HL 16851.) 19. Arteriolar Blood Flow Autoregulation in Sartorius Muscle. B. J. LALONE, AND P. C. JOHNSON, Department of Physiology, University of Arizona, Tucson, Arizona 85724. An earlier study of flow autoregulation in microvessels of isolated sartorius muscle (Johnson and Intaglietta, Physiologist l&266,1975) demonstrated that local reduction of arterial pressure commonly elicited increased arteriolar volume flow (calculated from vessel internal diameter and red cell velocity) as well as increased red cell velocity in some capillary networks. To determine if these responses are representative of the majority of the sartorius vasculature we have further examined blood flow autoregulation by simultaneous measurement of vessel internal diameter and red cell velocity in major distribution arterioles of sartorius muscle. Data were obtained from arterioles (initial i.d. : 45-l 30 pm) near their origin from the femoral artery during graded local reduction of arterial pressure from 100 to 40 mm Hg. About 75 % of the vessels studied showed an increase in calculated volume flow with arterial pressure reduction. The increase in calculated volume flow that occurred with arterial pressure reduction was usually associated with an increase in vessel diameter and a slight reduction in cell velocity. In some instances, however, arteriole internal diameter remained constant and red cell velocity increased with arterial pressure reduction, Since each of the arterioles studied delivered between 6 to 11% of the calculated gross flow to these muscles, these findings argue against a strictly flowdependent mechanism for blood flow autoregulation in sartorius muscle. (Supported by NJH Grants Nos. HL 15390, HL 05884, HL 17421.) in Alveolar Capillavies of Dog Lung Induced by High Perfusion Pressure. J. S. LEE, AND L. P. LEE,Division ofBiomedical Engineering, University of Virginia, Charlottesville, Virginia 22901.

20. Filtration

This study is aimed to investigate the filtration in individual subpleural alveoli and the factors specifying the initiation of alveolar flooding. A microscope-video system was used to measure the

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thickening of the alveolar septa and the flooding of alveoli. The filtration rate during the initial slower phase, Js, was found to be a constant ranging from 0.00024 to 0.006 ,um/secwith an average of 0.0013 Rm/sec. For the nine lobes studied, the logarithm of Js is distributed in a normal fashion, The filtration rate during flooding, Jf, ranges from 0.011 to 0.079 Bm/sec. The time between the start of perfusion and the initiation of flooding is distributed from 4 to 270 min. (The time of initiation is inversely proportional to Js.) On the other hand, JflJs is 19 + 7, and the percentage of area reduction of the alveoli at which flooding just initiates is about 79 + 9 %. These observations suggest that (1) when the thickness of the alveolar membrane doubles its initial value, damage is done by the high interstitial fluid pressure on the endothelium and epithelium to induce large filtration and hence flooding, and (2) Jf is about 20 Js. Based on these simple criteria, a filtration mode1 is constructed to integrate the edema development in individual alveoli with the weight gain of entire lobe. (Supported in part by grants from NHLI, Nos. HL 16812, HL 17108, and K04 HL 00004.) 21. The Pulmonary Arteriole. ROBERTAG. LINDAL, SOLBERNICK,AND SIDNEYS. SOBIN,University of Southern California School of Medicine, Los Angeles, California 90033. The immediate juxta-alveolar pulmonary microvessels of the cat (2 days to 18 yr) were examined by light microscopy with special stains to determine the validity of the term arteriole for lung vessels. The absence of smooth muscle in such vessels is current belief. In 2-day-old cat, arterioles of various types are found: fetal, with well defined media and round/blunt smooth muscle nuclei; early maturation, defined media with elongating smooth muscle nuclei and very thin internal and external elastica; maturation, in a few arterioles with a thin media there is a strong periodic acid Schiff (PAS) positive wail and elongated, darkly stained nuclei. Venules are strikingly different with total absence of smooth muscle layer. Up to 28 days, there is progressive qualitative change with increased attenuation of PAS positive media, increasing elongation of darkly staining nuclei, and increasing proportion of arterioles showing maturation changes. In 3f-month-old cat, and in adult cat, dog, monkey, and human, all arterioles show a highly attenuated PAS positive wall and elongated, deeply staining, tapered smooth muscle nuclei. Venules of similar dimension contain no muscle. These studies indicate that arterioles of the fetal-type undergo rapid maturation postnatally (possibly programmed) and persist in attenuated form throughout life. (Supported in part by NIH Grant No. HL 11152-10.) 22. Comparison of Pressure and F/o w Distributions in Skeletal Muscle and Splanchnic Microvasculatures. H. H. LIPOWSKY,ANDB. W. ZWEIFACH,AMES/Bioengineering, University of California, San Diego, La Jolla, California 92093. Intravascular pressures and volumetric flow rates have been measured throughout the successive arterial-venous segments of omentum (Rabbit), mesentery (Cat), spino-trapezius (Rat), and tenuissimus (Cat) preparations. For these distributions, a comparison of the gradients (slopes) of pressure and Sow through the microvessel hierarchy from artery to vein has been made, as well as the relative changes in the total intravascular cross-sectional area available for flow. From these data, we have also computed a segmental resistance to flow based on the pressure differential between segments and local flow rate. The data serve to elucidate the varied means by which arterial perfusion pressure is reduced in each successivearterial segment to provide a capillary pressure corresponding to the demands of the individual tissue in the normal flow state. This is accomplished by a deployment of serial and parallel microvessels characteristic of the specific tissue. The magnitude of the pre- to postcapillary resistance ratio was found to vary accordingly and directly reflect the spatial disposition and branching pattern of the microvascular network under steady state conditions. (Supported by Grant No. HL 10881, USPHS.) 23. Influence of Tissue Metaboiites and Oxygen Tension on Responsesqf Arterioles to Occlusion. J. H. LOMBARD,ANDB. R. DULINC, Department of Physiology, University of Virginia, School of Medicine, Charlottesville, Virginia 22901. Golden hamsters were anesthetized with Nembutal (60 mg/kg ip), and the cheek pouches were suffused with Ringer’s solution equilibrated with 0% OL (PO, = 9 + 2 mm Hg) or with 10% O2

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(PO, = 74 + 3 mm Hg). Microvascular diameters were measured during I-min occlusions of either single arterioles (MVO) or the whole vascular bed (WPO). Microvessel occlusion induced dilations which were significantly greater during 0% OZ suffusion than during 10% O2 suffusion. With whole pouch occlusion, large dilations occurred during both 0% and 10% suffusion. Recovery times were similar following release of MVO (both 0% and 10% 0, suffusion) and WPO (10% 0, suffusion); however, recovery from WPO (0% 0, suffusion) was significantly slower. PO, was measured with microelectrodes on the arterioles and in the tissue to assess 0, availability to the vascular smooth muscle (VSM) and parenchymal cells. During 0 % O2 suffusion, tissue PO, decreased to 5 + 2 mm Hg (MVO) and 11 f 1 mm Hg (WPO). On release of WPO, perivascular POZ recovered rapidly (tliz = 8 & 2 set), while recovery of arteriolar diameters as much slower (f,,* = 31 f 4 set). With 10% O2 suffusion, tissue, and periarteriolar PO, remained high during occlusion but marked dilation occurred, especially during WPO. Our data indicate that dilation is not due solely to effects of O2 deficiency at the VSM, but suggest a primary role for vasoactive metabolites. (Supported by NIH Fellowship No. HL-05026 and NIH Grant No. HL-12792.) 24. Temporal Variations in Microuascular Blood Flow. H. N. MAYROVITZ, R. F. TUMA, AND M. P. WIEDEMAN,Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140. Knowledge of variations in blood flow within a terminal vascular bed could help clarify both function and control of each of its several branching orders. Because of the difficulty in determining blood flow in consecutive branching orders simultaneously, sequential measurements are necessary. The value of this method depends on the degree of stability of flow. In order to study the temporal variation in flow, blood velocity and vessel diameter have been measured in first-order arterial branches of the bat wing, and these determinations have been used to calculate blood flow. The data obtained at a single arterial site were divided into IO-set intervals, and the average velocity within these intervals was calculated. From these determinations histograms were used to evaluate the variability in flow in a given animal over lo-min periods. Data so obtained in eight animals provided an overall index of flow variability. In general, velocity changes of three types were noted: (1) those synchronous with heart rate which produced periodic variations; (2) nonrhythmical changes due to undetermined mechanisms; and (3) those due to changes in vessel diameter. The range of velocities and flows were from 2.4 to 15.2 mm set-’ and 1.26 to 4.6 x lOm6cm3 set-‘. The composite analysis showed that an average of 85 % of the flow values were within k15 % of the mean flow for a lo-min period, thus indicating that it is possible to obtain reliable flow distribution data using sequential measurements. 25. Flow Behavior of ATP Depleted Erythrocyfes: Morphology-Rheology Relations. HERBERTJ. MEISELMAN,Department of Physiology, University of Southern California School of Medicine, Los Angeles, California 90033. Earlier reports of increased blood viscosity and greatly increased RBC membrane rigidity consequent to ATP depletion (e.g., Weed et al. J. Clin. Invest. 48,795,1969) have been followed by studies demonstrating minimal or nonexistent alterations in the mechanical properties of depleted RBC membranes; the present experimental program was initiated to examine the influence of cellular morphology on the flow behavior of suspensions of fresh and ATP-depleted human RBC. ATP depletion via 24-hr incubation at 37” caused: (1) RBC crenation (discocyte-echinocyte); (2) increased low shear rate viscosity; (3) enhanced non-Newtonian flow behavior. Virtually identical flow data were obtained with fresh ATP-rich RBC which were crenated via 2,4-dinitrophenol (DNP); shape change and viscometric behavior were dose-related up to 5 mM DNP. Both ATP-depleted and fresh DNP-treated cells could be returned to near-normal morphology and to normal (i.e., fresh control) flow behavior with the addition of the stomatocytic agent chlorpromazine hydrochloride (1-2 x 1O-5 M). These morphology-rheology results thus offer an alternative interpretation of the earlier viscometric data and suggest that decreased in uivo cell survival and disturbed microcirculatory flow associated with ATP-poor RBC are related to cell shape and extrinsic cellular deformability. (Supported by NIH Research Grant No. HL-15722 and NIH Research Career Development Award No. HL70569.)

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26. Efects of Hydrostatic Height on Pulmonary Lymph Protein Concentration. LEROY S. MOLSTAD, AND AIJBREY E. TAYLOR, Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi 39216. At different lung levels, the tissue Starling forces may be quite different. The tissue colloid osmotic pressure may become greater and/or tissue pressure may be less at the top of the lung, in order to counterbalance the decrease in capillary pressure. This study evaluates the lymph fluid draining upper and lower lobes of dog lungs that were isolated and perfused in either Zone III or Zones II and III conditions. Before bleeding the animals, in order to obtain blood for the perfusing system, both kidneys and adrenals were tied off, since the pulmonary resistance of the isolated lung was much less using shed blood from animals prepared in this fashion. The lungs were respired with warm humidified gas (95 % O2 + 5 % COz) at an average airway pressure of 2 mm Hg. Small lymphatics draining either upper, middle, or lower lobes were visualized 1 hr after isolating the lungs that had been placed in a vertical position. In five lungs at approximately 2-3 hr following isolation, we were able to obtain fluid from lymphatics draining regions of the lung that differed in height by 7-10 cm. In most instances, the colloid osmotic pressure of the lymph was similar at each lung level demonstrating no gradient between top and bottom of the lung. In two experiments, a difference in colloid osmotic pressure of 2 and 3 cm HZ0 was observed at a lung height difference of 10 and 9 cm, respectively. Although these are preliminary studies, we feel that no large difference exists in lymph plasma proteins between upper andlowerlunglevels,suggestingthatadifferenceincapillarypermeabilityoftissue hydrostaticpressures may exist between the top and bottom of lungs. (Supported by Grant No. HL 11477.) 27. Comparative Rheology of Erythrocyte SuspensionsFlowing in Small Bore Glass Tubes. J. S. MOSLEY, AND R. JENDRUCKO, Division of Bioengineering, Texas A&M University, College Station, Texas 77843. Suspensions of human, dog, and goat erythrocytes in normal saline and plasma were drawn in steady flow through straight lengths of microbore tubes by application of vacuum. For all suspensions studied, it was found that the logarithm of the wall shear stress was linearly related to the logarithm of the reduced flow velocity, Ofor 100 < D < 1000 set-‘. For flow in a 97 pm id. tube (0= 300 set-‘) with a feed hematocrit of 40 %, the apparent viscosities, bu.,,, of suspensions of erythrocytes in normally saline were found to be: goat, 3.0 cp; human, 2.5 cp; dog, 2.0 cp. Therefore papr,does not appear to be a simple function of erythrocyte geometry since both mean cell volume and diameter are ranked as follows: human z dog > goat. Preliminary measurement of the tube hematocrits by microcentrifugation of microbore tubes suggests that the observed differences in the apparent viscosities may be due in part to variations among species in the reductions in tube hematocrits due to the Fahraeus Effect. It was also found that for a feed hematocrit of 40 % with D = 300 set-i suspensions of erythrocytes in plasma exhibited a significantly greater apparent viscosity (papp= 3.6 cp) than suspensions of cells in saline (fiapp= 2.5). Since it has been shown that the tube hematocrit reduction associated with the Fahraeus Effect is identical for suspensions in plasma and saline (Microvascular Res. 6, 616, 1971), these results suggest that the observed differences in the flow properties are due solely to the nature of the suspending phase. 28. An Analytic Study of the Influence of Capillary Permeability and Axial Pressure Drop on Glomerular Ultrafiltration. H. D. PAPENFUSS, AND J. F. GROSS, Department of Chemical Engineering, University of Arizona, Tucson, Arizona 85721. A mathematical model has been developed to study the influence of the capillary wall permeability and the axial pressure drop on glomerular ultrafiltration. This analysis is an extension of the model proposed by Huss, Marsh, and Kalaba (Ann. Biomed. Eng. 3, 72, 1975) and studies the behavior of a typical glomerular capillary in the rat. The governing equations for the axial and radial transport of fluid have been derived with the latter based on Starling’s hypothesis. The solution of the nonlinear, coupled differential equations provides predictions of the axial volume flow, the hydrostatic pressure, the plasma protein concentration, the hematocrit, and the filtration rate along the capillary. The hydrostatic pressure, concentration, and hematocrit at the afferent arteriolar end, the wall permeability,

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the extravascular pressure, and the pressure at the efferent arteriolar end must be specified. Typical values of these parameters that are based on experimental data obtained on rats have been selected for this study. An important result of this analysis is to show that in contrast to previous work an increase of the axial volume flow by means of enhancing the axial pressure drop is not necessarily coupled with an increase of the filtration rate. Instead, as the axial pressure drop increases, the filtration rate approaches a maximum whose value depends on the wall permeability, and then declines with a continuing increase in axial flow. In this regime the filtration rate can be increased only by augmenting the wall permeability. (Supported by PHS-HL Grant No. 17421.) Vein: An Unusual Vasomotor Mechanism. B. L. PEGRAM,R. D. BEVAN, AND J. A. BEVAN,Department of Pharmacology, School of Medicine, University of California, Los Angeles, Los Angeles, California 90024. The buccal segment of the rabbit facial vein develops tone spontaneously in eitro. It relaxes in response to norepinephrine and transmural nerve stimulation, and this biphasic response is reversed by propranolol (10m6M). Histamine causes vasoconstriction exclusively. Phenoxybenzamine (10e5 M) increases the size of the neurogenic relaxation and displaces the dose-relaxation curve to the left. The response to nerve activation is obliterated by tetrodotoxin, chronic bilateral superior cervical ganglionectomy, and reserpinization. Histological and histochemical examination show a vessel with a poorly demarcated tunica adventitia and tunica media, the latter consisting of five to seven layers of vascular smooth muscle cells orientated mainly in a circular direction. No elastin was seen and randomly orientated collagen fibers separated the media from the intima. A dense adrenergic plexus ramified throughout the muscle layer. These features are consistent with a vessel which maintains intrinsic tone in vitro, in which the response to adrenergic nerve activation is vasodilation mediated by /?-adrenergic receptors. (Supported by USPHS Grant No. HL 8359.) 29. Rabbit Facial

Corrections to Crone UpsIope Permeability Method. W. PERL, Departments of Medicine and Physiology, New Jersey Medical School, Newark, New Jersey 07103. The Crone (1963) method of deducing endothelial permeability from single injection indicator curves is to determine extraction E as 1 minus an average ratio of test solute (concentration/dose) to vascular solute (concentration/dose) on the upslope portion of the indicator curves. Thence E = E= 1 exp(-a), where a = PS/F (permeability x surface/water flow). A simple back diffusion correction method (A) is to use a modified Kety (1951)-Renkin (1959) equation E = eexp(-kt), where k = &F/(V- yV,) and V, = vascular volume, V= total volume of distribution, y= E/U and t= VI/F= vascular mean transit time. An alternative method (B) is to set exp(-kt) = (V - VI )/( V - y V,), which satisfies overall extravascular solute balance from t = 0 to m and reduces correctly in the flow limit a = 00,e = 1, y = 0 to 1 - E, = VI/V = instantaneous equilibration of test solute between its vascular and extravascular volumes of distribution. For dog lung endothelium (Per1 et al., Amer. J. Physiol., in press), (A) increases the Crone permeability by 7% (sodium ion) to 76% (valeramide). (B) gives about half these increases. Tritiated water permeability is 180 x IO-’ cm/set by (A) and 110 x lO-5 cm/set by (B), with VI/V obtained from the simultaneously measured octanediol upslope, assumed flow-limited. In general, (A) or (B) yields PS of an indicator x from the ratio of its upslope (concentration/dose) w, to that of an indicator y of known PSand, for both indicators, the known (not necessarily equal) ratio of total to vascular volumes of distribution; thus, by (B), w,/w, = [1 - E,(V, - VJ (V, - yx V,,)]/[l - E~(V, - V,+)/(V, - yy VJI. (Supported by Grants Nos. HL-12974 and HL-12879 from NHLI.) 30. Back Diffusion

3 1. Basis for a Spectrophotometric Methodfor Blood Oxygen Saturation Determination in the Terminal Microvasculature. ROLAND N. PITTMAN, Department of Physiology, Medical College of Virginia,

Richmond, Virginia 23298. We have previously presented a microspectrophotometric method to determine blood O2 saturation in microvessels (20-100 pm) using green wavelengths (J. Appl. Physiol. 38, 321, 1975). The extension of the method to smaller vessels (5-20 pm) requires a larger red cell (RBC) hemoglobin (Hb and HbOz)

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absorbance signal than can be obtained using the green wavelengths. The Hb and HbOz absorption spectra suggests that a sufficient sensitivity to changes in 0, saturation can be obtained in the blue spectral region (near the Soret band). The transmitted light intensity through whole blood depends on both hemoglobin absorption and RBC light scattering. The scattering contribution depends on the refractive indices of Hb(no) and HbO&) which vary rapidly with wavelength near the Soret band. Also, no and nI are different over part of this spectral region. Thus, the scattering contribution to the transmitted light intensity can depend on O2 saturation, and it is necessary to choose wavelengths as close as possible to isorefractive ones. Spectra were obtained for Hb and Hb02 solutions and RBC suspensions. The corresponding solution and suspension spectra were found to be linearly related over a restricted wavelength range containing two isosbestic wavelengths and several at which there are large differences in Hb and HbO, extinction. An identical linear relation was found between fractional HbO, and an optical density ratio for hemoglobin solutions and RBC suspensions (corrected for scattering). Preliminary measurements have been made on terminal microvessels in the hamster cheek pouch with a video microdensitometer. (Supported by AHA 75 7 14.) 32. Effects of Tobacco Smoke Exposure on the Response of Capillary

Blood Flow to Norepinephrine.

DANIEL RICHARDSON,Departments of Physiology and Biophysics, University of Kentucky College of Medicine, Lexington, Kentucky 40506. The purpose of these studies was to test the hypothesis that tobacco smoking potentiates the effects of catecholamines on capillary flow dynamics. One group of rats was exposed daily to tobacco smoke from five reference cigarettes for a period of 5 to 8 months, while a second group was sham treated. At the end of the treatment period, RBC velocity in selected capillaries of the mesentery was measured by the dual-slit photometric method before and during topical suffusion of graded levels of norepinephrine (NE) ranging from 0.4 to I .O pg/ml. With the majority of the vessels studied in both groups, NE elicited an all-or-none response in which RBC velocity showed only moderate variation from control as NE concentration was increased until a point was reached at which complete flow arrest occurred. However, at each concentration tested the smoke exposed animals showed an increase in the number of capillaries in which flow arrest was elicited during suffusion. The results suggest that this was due to an increased incidence of formed element plugging not a higher sensitivity of the vessels per se to NE. It is concluded from these studies that tobacco smoking does modify the effects of NE on capillary blood flow, but the mechanism seemsto he related to changes in the blood itself, not variations in vascular sensitivity to catecholamines. (Supported by University of Kentucky Tobacco and Health Research Institute, Contract No. 24016.) 33. Experimental Test of Multisolute Corrections to Observed Reflection Coefficients in Dog Lung Endothelium. A. B. RITTER, Department of Chemical Engineering, Stevens Institute of Technology,

Hoboken, New Jersey 07030 and V. MINAK AND W. PERL, Department of Medicine, New Jersey Medical School, Newark, New Jersey 07103. In isolated, perfused dog lung, the “observed” reflection-filtration product, (oP~S),,~~,of the bloodtissue (endothelial) barrier for a test solute was determined as the initial rate of weight change of the lung per step function increment of osmolal concentration of test solute in the perfusate (Vargas and Johnson, 1964). The observed filtration coefficient, (P,,!%,, was determined in the same preparation (Pappenheimer and Soto-Rivera, 1948).The observed reflection coefficient, e,,,,% (solute), was calculated Osmotic buffering corrections of cobsto obtain a true o may be substantial as (uPfShl(~fShbS. (Grabowski, 1974; Perl, Choudhury, and Chinard, 1975). Accordingly, we varied both perfusate and test solute compositions. Results for uoohs _+ SD (n) are: “NaCl” (Ringer) perfusate 0.08 & 0.06 (43); NaCl in “sucrose” (90% sucrose - 10% NaCl Ringer) perfusate 0.09 f 0.01 (5); sucrose in “NaCl” 0.11 f 0.05 (14); sucrose in “sucrose” 0.08 + 0.03 (9). The individual filtration coefficients used to determine crohswere averaged to give P,S/SOO = 0.065 + 0.04 (35) cm set-‘. The differences are not statistically significant, but the data tentatively suggest that (a) osmotic buffering and multisolute (solute-solvent-solute) effects are greater with sucrose perfusate than wtth NaC1 perfusate; (b) true c (sucrose) > true 0 (NaCI); and (c) the preceding absolute values of cobsmay be underestimated because of flow limitation. (Supported by NHLI Grant No. HL-12879.)

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34. Red Cell Velocity and Plasma Transit Time in the Cerebral Microcirculation of Spherocytic Mice. WILLIAM I. ROSENBLUM, Medical College of Virginia, Richmond, Virginia 23298.

127 Deer

Spherocytosis is a hyperviscosity syndrome where increased RBC rigidity produces the rheologic abnormality. Spherocytic deer mice provide a model of human spherocytosis. In the present study, we observed arterioles and venules on the cerebral surface of 15 spherocytics and utilized high speed microcinematography to measure RBC velocity in individual vessels and low speed microcinematography to measure fluorescein transit time from arteriole to venule. Eighty-one measurements were made in arterioles and venules 1346 pm in i.d. RBC velocity was the same as that observed in nonspherocytic deer mice, ranging from 2-26 mmjsec at the vessel center and from l-l 7 mmjsec at the wail. Fluorescein transit time was 0.67 f 0.21 set in controls and 0.48 f 0.25 set in spherocytics (P < 0.05). Spherocytics had lower HCt than controls (29 f 5 vs 35 + 5; P < 0.02). Blood viscosity measurements at shear rates of 46 to 230 set-’ showed that the low HCt offset the rheologic effect of rigid RBC; viscosity of anemic spherocytics was elevated only in relation to that of normals with HCt reduced to spherocytic levels, but not in relation to that of nonanemic normals. Previous work shows that reduced HCt should be accompanied by accelerations of both RBC and plasma. Since plasma (i.e., Fluorescein) was accelerated in the spherocytic mice, we conclude that increased RBC rigidity had little or no effect on plasma movement, but did reduce RBC velocity, preventing anemia from accelerating the RBC. The data provide a striking contrast with our earlier studies of macroglobulinemia or polycythemia, since in the latter hyperviscous diseases, the rheologic abnormalities result in a greater effect upon plasma transit than upon RBC velocity. (Supported by Grant No. HL 12775 and John A. Hartford Foundation.) 35. A Mass Transport Modelfor Predicting O2 Distribution in the Autoregulating Myocardium. Rev W. SCHUBERT, AND W. J. WHALEN, St. Vincent Charity Hospital and Case Western Reserve University,

Cleveland, Ohio 44115. Recently we concluded that the existence of hypoxic areas (p02 < 5 mm Hg) in the autoregulating myocardium seemed to be an essential feature of autoregulation (Physiologist 18. 382, 1975). We hypothetized that they may have a role in the control of O1 transport to the heart. These ideas are being investigated by development of an 0, mass balance model for the whole heart based on a repeating histological tissue-capillary structure. We sought a simple tissue model capable of displaying 0, histograms similar to those we measured. Models involving solution by simulation were not considered since repeated evaluations, needed in the total model, are costly. The Krogh cylinder allows only radial gradients and transport. The Krogh-Blum model allows radial and axial gradients but transport only by the former. Histograms from both were unsatisfactory. Michaelis-Menten kinetics improved the O-15 mm Hg range only. Results from the last model indicated that the axial gradients were much larger than radial, suggesting that we assume the radialdirection as “well mixed,” with radial transport by way of an effective capillary permeability and axial transport by diffusion. This formulation allows for a uniqlre interpretation of the 0, histogram as the reciprocal of the axial 0, gradient. A constrained nonlinear least squares treatment of the histograms was used to estimate model parameters, the constraints being derived from our physiological data. This model was superior to all others in representing the essential features of the histograms we obtained in the Krebs-Hensleit perfused heart, and we plan to include it in the total model. (Supported by Grants Nos. HL 11906and HL 52718 from USPHS.) 36. The Filtration Coefficient of the Rat Glomerulus as Deduced.from a Stereological Study of its Filtration Surfaces. S. M. SHEA, AND A. B. MORRISON, Department of Pathology, CMDNJ-Rutgers

Medical School, Piscataway, New Jersey 08854. It has been suggested, because of the porous substructure of the glomerular slit diaphragm (GSD), that the glomerular filter may consist of two filtration surfaces in series, the peripheral glomerular basement membrane (BM) and peripheral GSD. Moreover, there is evidence that the BM is more restrictive of macromolecules than the GSD during ultrafiltration (J. Cell. Biol. 63, 883, 1974; Fed. Proc. 34, 877, 1975). Accordingly, we have measured morphometrically the length of the peripheral

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glomerular slit and the area of the peripheral BM from electron micrographs of rat kidney tissue. The slit length per 60,000 glomeruli was 29.2 f 1.7 km (corresponding to a pore area of 2.96 cm*), and the BM area was 55.3 & 3.3 cm* (corresponding to a pore area of 44.24 cm’). If the GSD pores are 40 x 140 8, rectangular cylinders, and the BM pores are treated as hydraulically equivalent to slits 30-A wide, and if the GSD and BM are, respectively, 70- and 1400-A thick, we obtain a filtration coefficient K, = 2.238 x 10m6cm3/(dynes.sec), or 0.050 nl/(mm Hg.sec) per glomerulus. Given the uncertainties in assumed parameters (especially in the pore size of the BM), this is in reasonably good agreement with the micropuncture estimates, respectively, of 0.059 nl/(mm Hg.sec) and 0.078 nl/(mm Hg.sec) per glomerulus obtained by Brenner et al. (Amer. J. Physiol. 223, 1184, 1971; J. C/in. Invest. 52,1500,1973). Accordingly, we suggest that the glomerular filter is bilaminar with distinct pore sizes, and that paradoxes in the use of a unilaminar isoporous model for physiological clearance studies (Biophys. J. 15, 887,1975) are to be expected. (Supported by USPHS Grant No. AM 13495.)

37. Intravascular Coagulation Caused by Circulating Immune Complexes. J. G. SIMPSON(Sponsor, J. A. G. RHODIN), Department of Pathology, University of Aberdeen, and Department of Anatomy,

University of Michigan 48109. It is generally assumed that the local blood coagulation associated with immune complex deposits has taken place as part of an in situ inflammatory reaction. This study was designed to investigate the systemic effects of immune complexes on the haemostatic mechanism. Circulating complexes were produced in rabbits by iv injection of antigen into sensitized animals or by iv injection of preformed complexes into nonsensitized animals. The antibody involved in complex formation was characterized as a complement-binding IgG immunoglobulin. A sensitive anticomplementary assay showed that peak complex formation occurred 5 min after injection, with rapid disappearance from the circulation. There were concomitant changes in blood coagulation and fibrinolysis, indicating intravascular coagulation and depletion of fibrinogen, platelets, factors V and XII; appearance of fibrin and altered platelets in the dynamic circulation; reactive fibrinolysis; and fibrin-platelet deposition in capillaries. The results indicate that local blood coagulation in immune complex diseasesmay be partly secondary to a systemic reaction.

TotalProtein in Lymph and “Secreted” Fluidin Cat Small Intestine. TAYLOR, AUBREY E., D. NEIL GRANGER,AND NICHOLASA. MORTILLARO,Department of Physiology, University of Mississippi Medical Center, Jackson, Mississippi 39216, and Department of Physiology, University of South Alabama, Mobile, Alabama 36688.

38. Analysisof

Lymph and “secreted” fluid were collected from five isolated, autoperfused cat ileal preparations at a venous pressure of 30 mm Hg. Colloid osmotic pressures of lymph and lymph flow were measured at S-min intervals, and the colloid osmotic pressure and flow of “secreted” fluid were measured at either 15- or 30-min intervals following elevation of venous pressure. The lymph to plasma protein concentration ratio (L/P) was0.55 f 0.16at acontrolvenouspressure of 1Omm Hg; followingelevation ofvenous pressure to 30 mm Hg, the L/P ratios responded in a biphasic fashion : the L/P ratios decreased to 0.06 & 0.016 before “secretion” began but increased to a higher value of 0.10 + 0.05 after fluid entered the intestinal lumen. The “secreted” fluid to lymph protein concentration ratio (S/L), which should be some indication of mucosal sieving, was approximately I at low “secretion” rates and decreased to approximately 0.2 as “secretion” became copious. These experiments indicate that intestinal capillaries have a maximum average reflection coefficient for plasma proteins of 0.92 i 0.97 and that the intestinal mucosa is at first very leaky to the proteins (a = 0) in a small portion of the mucosal membrane, and that molecular sieving occurs at higher “secretion” rates such that intestinai “tissue” proteins are actually concentrated above the minimal “tissue” values. These findings are in agreement with the earlier studies of Yablonski (Dissertation, University of Minnesota, 1973, and J. Clin. Invest., in press) using an isolated dog intestinal preparation. (Supported by Grant No. HL 15680.)

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39. Changes in Skeletal Muscle Blood Flow Following Mechanical Stimulation. R. F. PUMA, K.-E. ARFORS,G. RUTILI, L. LINDBOM,AND M. CHILDS, Department of Experimental Medicine, Pharmacia A.B., Uppsala, Sweden, and Temple University School of Medicine, Department of Physiology, Philadelphia, Pennsylvania 19140. The effect of trauma resulting from the preparation of a microvascular bed for observation may in part account for the discrepancies between micro- and macroflow measurements, and also may explain some of the discrepancies between the observations of various investigators studying the same tissue. In this investigation, the effect of mechanical stimulation causing a mild trauma, similar to that which may be incurred during surgical preparation, on blood flow in the tenuissiumus muscle was studied. Both total muscle blood flow and individual capillary RBC velocities were measured. Total muscle blood flow was measured through the use of radioactive microspheres. After careful preparation of the muscle for observation, a 30-min equilibration period was allowed to pass. Control flow measurements were then taken using these techniques. The muscle was then stretched and stroked with a blunt metal probe. Following this trauma, the flow measurements were again repeated. Both measurement techniques demonstrated a significant increase in skeletal muscle blood flow following mild mechanical trauma. Capillary RBC velocity increased two to ten times, and total muscle blood flow increased five to ten times following the trauma. Therefore, it can be seen that mild surgical trauma may result in skeletal muscle blood flow, which is higher than that present under normal resting physiological conditions. These results show that the muscle is extremely sensitive to mechanical trauma and that great care must be taken during the preparation for microvascular observation. 40. Exclusion Effects in Macromolecular Solutions. P. D. WATSON, AND F. S. GRODINS (Sponsor E. M. RENKIN), Department of Human Physiology, University of California, Davis, California, and Department of Biomedical Engineering, University of Southern California, Los Angeles, California. In 1958 Ogston reported an analysis of the volume excluding effect of a three-dimensional array of straight rods (Trans. Faraday Sot. 54, 1754,1958) which he and others have applied to macromolecular solution, including hyaluronic acid (HA). It is shown that this model can quantitatively explain the osmotic pressure data of HA-albumin solutions reported by Wiederhielm (“Biomechanics,” Fung et al., eds. p. 273,1972). If K,, is the fraction of the solution volume available to the albumin, obtained from Ogston’s analysis, the apparent increased concentration of albumin C’ is given by C’ = C/K,,, where C is the original concentration. Combining this with the two polynomials for the osmotic pressure of HA and albumin solutions given by Wiederhielm, the total osmotic pressure can be calculated. One parameter, a factor multiplying the rod concentration, was adjusted to obtain a good fit. A two-phase system, incorporating Ogston’s result in a gel phase containing HA and a free-fluid phase with no HA, was used with the in vivo volumes of distribution data for dextrans obtained by Garlick and Renkin (Amer. J. Physiol. 219,1595,1970). When adjusted to fit this data, the model predicts HA concentrations in the gel phase around 0.2 g % and a free-fluid fraction of the total volume between 0 and 0.5. (Supported by NIH Grants Nos. GM-531 10 and HL-05135.) 41. Intrinsic, Viscoelastie Properties of Erythrocyte Membranes of Dlyerent Vertebrate Animals. RICHARD WAUGH, AND EVAN EVANS, Department of Biomedical Engineering, Duke University, Durham, North Carolina 27706. Using equilibrium and relaxation measurements of micropipet-aspirated membrane projections, the membrane surface “shear” moduli and surface “viscosity” coefficients were obtained for the plasma membranes of selected animals from five vertebrate classes. These coefficients characterize the first order, recoverable material behavior for the membrane as a viscoelastic solid. Three types of nucleated cells (amphibian, avian, and “boney” fish) exhibited similar hyperelasticity with “shear” moduli of 7-8 x lo-’ dynes/cm for “stretch” ratios up to 3: 1 (finite strains of the order 400%); the surface viscosity coefficients were also similar for amphibians and avians, 6-7 x 10m3dyne-set/cm (no viscosity data was obtained for fish erythrocyte membranes). The properties of nucleated cell membranes for reptiles were higher; the “shear” moduli were in the range 2-3 x 10-l dynes/cm, and the surface viscosity, 4-10 x lo-’ dyne-set/cm. In contrast with the nucleated erythrocyte membranes, membranes

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of nonnucleated, mammalian cells all exhibited lower “shear” moduli, 7-10 x 10e3 dynes/cm, and surface viscosity coefficients, ~10~~ dyne-set/cm. The pressure versus length of aspirated membrane projections of both nucleated and nonnucleated cells correlated well with the analysis of a twodimensional, viscoelastic membrane in large deformation (at constant area). The particular “donor” animals included: amphiuma, frog, turkey, toad fish, turtle, iguana, opossum, tree-shrew, and human. (Supported by NSF Grant No. GK 43118.) in Rat Mesenteric Membrane. HAROLD WAYLAND, AND JAMESR. Fox. Division of Engineering and Applied Science, California Institute of Technology, Pasadena, California 91125.

42. Serum Albumin Difision

The ease of passage of macromolecules across the mesentery mesothelium (W. G. Frasher, personal communication) has led us to reexamine our measurements of the apparent diffusion coefficient of serum albumin in the mesenteric membrane. Tracer concentration profiles were obtained in the rat mesentery by video-fluorimetry of rat serum albumin tagged with fluorescein isothiocyanate (Wayland and Fox, Abstract 120, 1st World Congress for Microcirculation, Toronto, Canada, 1975). When both sides of the mesenteric membrane were in contact with mineral oil (warmed to 37”), values for the apparent diffusion coefficient ranged from 2-2.5 x lo-’ cm’/sec. This is in contrast to values of 47 x lo-’ cm2/secreported for rat and cat when the membrane was exposed to an aqueous physiological solution (Wayland and Fox, 1975; Nakamura and Wayland, Microvase. Res. 9, l-20, 1975). These new values obtained where transmesothelial transport is minimized agree with values reported by Granger (Abstract I 15, 1st World Congress for Microcirculation, Toronto, Canada, 1975) for serum albumin diffusion coefficients in normally hydrated human umbilical cord. (Supported by USPHS Grant No. HL08977 and a grant from the John A. Hartford Foundation.) 43. Platelet Aggregates in Denervated Arteries, MARY P. WIEDEMAN,AND RONALDF. TUMA, Depart-

ment of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140. In ciuo microscopic observation of experimentally produced platelet aggregates in the vascular bed permits evaluation of ways to inhibit or enhance aggregation and adherence to the vessel wall. The role of sympathetic vasoconstrictor nerves in the response of platelets to vessel wall changes occurring after delivery of a laser beam was studied by comparing aggregation and adherence in innervated and denervated vessels. The length of time that an aggregate continued to build up and remain fixed to the wall was used as a measure of platelet activity. When embolization and growth of the aggregate ceased, thereaction ofplatelets to the initial stimulus was considered to be over. Platelet activity was diminished in surgically denervated vessels compared to innervated vessels. Chemical denervation with phenoxybenzamine also resulted in a dimunition of platelet activity. Conversely, platelet activity could be prolonged by intra-arterial infusion of concentrations of epinephrine too weak to produce vasoconstriction. Other vasoactive drugs, such as dipyridamole, will be tested for platelet inhibition.

44. Cremasteric Arteriolar Response to Drugs by Topical atld Intra-Arterial Route of Administration in the Mouse. TAKASHI YAMAKI, SILVIOBAEZ, AND LOUISR. ORKIN, AECOM, Bronx, N.Y. 10461.

Previous reports have demonstrated a greater drug threshold response of arterioles (art]) to intraarterial (i.a.) than to topical (T) epinephrine in an isolated vascular model (Angiology 12, 452, 1964). This report concerns similar determination of response in the intact cremasteric microvasculature in the mouse, using norepinephrine (n. epin.), vasopressin (vasopr.), and angiotensin (angiot.) as the stimulating drugs. Male mice 36.2 + 1.94 gm (n, 19) was used in all experiments. Under pentobarbital anesthesia (30 mg/kg, im), the left femoral artery was cannulated and the ipsilateral cremaster muscle was prepared by standard procedures for microscopic observation and measurement. Change in arteriolar diameter were measured by image-splitting methods as the same amount and volume of the drug solutions were delivered T and then i.a. With all three agents a greater arteriolar constriction was obtained by the topical route. The diameter of 22 target artl. was mean + SD, 15.6 + 3.69. The concentration of n. epin. was 0.14 + 0.317, of angiot. was 48 + 0.280 pg/ml, and of vasopr. was

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0.03 &-0.067 i.u./ml. P < 0.001 for all three drugs. A greater response by intra-arterial than by topical route of administration of three vasoactive drugs was extended and confirmed in striated muscle microcirculation in mice. The mechanism of the difference in response remains unresolved. (Work supported by Grant No. NIH HL-06736.) TABLE VASOCONSTRICTIVE

Results top i.a.

RESPONSE

N. Epin. (n, 22)

Vasopr. (n, 14)

Angiot. (n, 12)

51.0 f 4.54 18.7 f 9.71

49.4 * 4.03 19.4 * 7.51

49.4 + 2.57 20.5 + 6.88

Microcirculation of the Frog. R. T. YEN, AND Y. C. AMES-Bioengineering, University of California, San Diego, La Jolla, California, 92093.

45. Resistance and Pressure Profiles in Pulmonary FUNG,

The distribution of pressure and resistance in various generations of blood vessels is of great interest to pulmonary circulation because, unlike peripheral circuits, the pulmonary capillaries are sites of major pressure drop. To study this problem, direct dynamic intravascular micropressure measurements of small intrapulmonary vessels and capillaries were performed by micropuncture technique. The resistance null-balance feedback servo system with a Wiederhielm type of probe was used. Rana Pipiens frogs were used for the experiments. By perfusing the lung either with saline or with blood from donor frogs through an isolated, arrested lung with controlled airway pressure, the following parameters were measured: the lung volume, the pulmonary artery pressure, pulmonary venous pressure, pulmonary blood flow, airway pressure, and the microvascular pressure. The interrelationship of these parameters was investigated. The results will be used to assessthe relationship between pulmonary microcirculation and macrocirculation. (Supported by IJSPHS Grant No. HL-12773 from NHLI.)