MICROVASCULAR

RESEARCH

43, 73-86 (1992)

Microvascular Flow Response to Localized Application of Norepinephrine on Capillaries in Rat and Frog Skeletal Muscle HANS HUBERT DIETRICH AND KAREL TYML’ Department of Medical Biophysics, University of Western Ontario; and The Robarts Research Institute, London, Ontario, Canada, N6A SC1 Received September 26, 1990

Recently, Dietrich (1989, Microvasc. Res. 38, 125-135) demonstrated that a local application of a minute amount of norepinephrine (NE, 5.5 mM, 0.01-88 pmole) on a capillary in rat mesentery can elicit constriction of the feeding arteriole 0.5-1.0 mm away. This constriction can reduce or even stop blood flow in capillaries supplied by the arteriole. The main objective here was to show that the phenomenon of reduced flow occurs not only in the rat mesentery but also in other tissues and species. We chose to study the rat tibialis anterior and frog sartorius muscles. Using the same intravital video-microscopic approach as in the mesentery, strong NE stimuli (3 mM) were applied iontophoretically 48 times to 19 capillaries in 10 rats anesthetized with pentobarbital. They resulted in significant reductions (average: 80%) of the red blood cell velocity (V,,,) in capillaries. The onset of these reductions (i.e, 10% decrease from control) occurred within 3-52 set (average: 20.9 set) from the time of NE application. Reductions lasted 6.0 min. The same stimuli were applied 42 times to 15 capillaries in 6 frogs anesthetized with urethane. The average V,,, reduction was 86%. The onset occurred within 30.6 set while the reduction lasted 6.6 min. Under the same conditions, arteriolar diameters in the sartorius muscle decreased significantly from 28.5 to 22.5 pm (n = 8). We also used local microinjection of small droplets of NE (30 mM) to 13 capillaries in 7 frogs. This resulted in a significant V,,, reduction of 64% with an onset time of 44.2 set and a reduction duration of 17.2 min. Weak NE stimuli (3 PM) applied iontophoretically to 10 capillaries in 5 frogs resulted in marginal, but significant, V,,, reductions (9%). The present study demonstrates that the phenomenon of reduced flow after local application of NE may be a general phenomenon as it occurs also in skeletal muscle in both rat and frog. Our accompanying paper addresses the hypothesis that the phenomenon reflects communication of a NE-induced signal along the capillary. o 1992 Academic

Press. Inc.

INTRODUCTION Recently, it has been demonstrated that a local vasoactive stimulus can be conducted along the arteriole (Segal et al., 1989). Segal and Duling (1989) proposed that this conduction is mediated via smooth muscle and/or endothelial cellto-cell communication. Our work focused on the question whether such a stimulus can also be conducted along the capillary. Dietrich and Weigelt (1986) and Dietrich (1989) showed that a local stimulation of a capillary in the rat mesentery with ’ To whom reprint requests should be addressed. 73 0026.2862/92 $3.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.

74

DIETRICH

AND

TYML

norepinephrine (NE) can be propagated in a retrograde manner to the supplying arteriole. Although these workers demonstrated the presence of adrenergic nerves near capillaries, they could not explain the retrograde communication entirely by the nerve conduction. As an alternative, endothelial cell-to-cell communication was proposed. The main objective of the present study was to show that the retrograde propagation is not limited only to rat mesentery, but that it also occurs in capillaries of other tissues and species. We chose to study the rat tibialis anterior and frog sartorius muscles. The rationale for using these muscles was the general concensus in the literature that adrenergic nerves are not found near capillaries in skeletal muscle (ohlen et al., 1988; Fleming et al., 1987). We reasoned that if the presence of the phenomenon is demonstrated in this tissue, then, in future studies addressing the mechanism of the phenomenon, the use of the skeletal muscle may yield evidence supporting the above alternative of endothelial involvement. A secondary objective of the present study was therefore to confirm the absence of adrenergic nerves near capillaries in the two chosen muscles.

MATERIALS

AND METHODS

Animal Preparation Rat tibialis anterior muscle. Ten female Wistar rats (average weight 192.4 ? 10.4 SD g) were anesthetized with pentobarbital (Somnotol, 65 mg/kg body wt). The animals were placed in a lateral position on a heating pad on the stage of an intravital microscope (ELR, Leitz). The rectal temperature was maintained at 37”. The left tibialis anterior (TA) muscle was exposed by cutting and reflecting both the skin and the overlying biceps femoris muscle. Using fine forceps, the connective tissue layer covering the TA was carefully removed and the muscle was covered by a plastic coverslip. A hole (3 mm in diameter) in the coverslip filled with liquid mineral oil allowed access to the muscle surface with micropipettes, while the oil prevented drying of the muscle. Humidified N2 was blown over the surface to prevent air from reaching the muscle surface. The microscope was equipped with long working distance lenses 4X/O. 12 N.A., 10X/0.2, and 20X/0.32. A sensitive video camera (MTI) scanned the microscopic image of the microcirculation and displayed it on a monitor (Panasonic WV 5410) with a final magnification ranging from 154X to 770X. Images were stored by means of a 3/4 in. recorder (Panasonic NV 9240XD) and analyzed off-line with a slow motion video recorder (Sony BVU 820). Frog sartorius muscle. Eighteen frogs (Rana pipiens, 49.3 + 27.7 SD g) were anesthetized by subcutaneous injection of urethane (20% in physiological saline, 0.4 g/100 g body wt.). Each frog was placed in the supine position on the stage of the Leitz microscope and a sartorius muscle was exposed by cutting and reflecting the overlying skin. The muscle was covered by a plastic coverslip with a hole to allow access to the muscle surface with micropipettes. The same microscopic and video equipment was used for visualization and recording of the microcirculation at the surface. In both muscles, blood vessels running in parallel to the muscle fibres were defined as capillaries. Vital microscopic observation of these vessels never showed

FLOW

RESPONSE

TO NOREPINEPHRINE

75

any sign of smooth muscle cells. For best visibility and accessibility with micropipettes, we chose capillaries with steady blood flow near the muscle surface. of NE Zontophoresis. Microelectrodes were pulled from blanks (Kwik Fill, 1 mm o.d. thin walled; WPI New Haven, CT) and filled with NE solution (NorepinephrineHCl in phosphate-buffered saline; Sigma, St. Louis, MO). The saline was used to achieve an isoosmotic condition in the pipette (Stone, 1985). The NE solution was stained with 0.5% Evans blue. Each microelectrode was connected to a direct current source that provided a positive current for ejection of the positively charged NE ion. An Ag/AgCl wire inserted in a hypodermic needle, used as a reference electrode, was placed subdermally in the hind limb. The current was measured by a picoammeter. During each application, the tip of the electrode (less than 1 pm in diameter) was positioned by a micromanipulator near the capillary of interest. The closure of the electric circuit upon contact with the muscle surface was indicated by the withdrawal of the stain (negatively charged) from the tip in the electrode. Reversing the current retained NE and ejected the stain. The phosphate-buffered saline stai4fed with 0.5% Evans blue was used as a control. There were two reasons for &sing Evans blue. The first was to check the patency of the electrode tip by observingejection and withdrawal of the stain from the tip. The second reason was to ascertain the absence (or presence) of a convective current at the oil-tissue fluid interface. This current could possibly transport NE from the capillary to the arteriole. By ejecting the dye, the presence of the current would be detected by a one-directional sweeping movement of the dye away from the electrode tip. In this study, Evans blue was not used as a tracer of the diffusion process. Since other studies used distilled water as a solvent for NE (Segal and Duling, 1989), we carried out several experiments in which saline was replaced by distilled water. Results from these experiments are reported separately. Microinjections. An alternative method of NE application to capillaries by microinjection was also used (Dietrich, 1989). Briefly, micropipettes were pulled from Pyrex glass blanks (Dow Corning, 1 mm o.d.) to a tip of about 4 pm in diameter. The pipettes were filled with liquid mineral oil and attached to a nanoliter pump (pump capacity 80 nl/min; Bachhofer, FRG). Norepinephrine-HCl (Sigma) was dissolved in phosphate-buffered saline and stained with Evans blue (0.5% w/v, Sigma). Small NE droplets were aspirated into the pipette. Using a micromanipulator, the pipette was then lowered into the oil layer within the hole of the coverslip and positioned above the capillary of interest. The drug was then ejected, forming a sphere attached to the tip. The diameter of the sphere was measured to estimate the amount of ejected drug. Next, the pipette with the sphere was lowered toward the capillary. The sphere burst when it reached the oil-tissue fluid interface and NE was deposited on the capillary. Stained droplets of phosphate-buffered saline were used as controls. Local Application

Histology

Rat and frog muscles were examined for adrenergic nerves using the same fluorescence technique as in our previous study of the rat mesentery (Dietrich, 1989). Briefly, frozen muscle sections (15-20 pm thick) were stained with glyoxylic

76

DIETRICH

vRBC[Iwn/sl

TYML

NE

-200-t -100

AND

.tTFR

-50

0

, TRPl,fTCO

100

,

150

200

FIG. 1. Time course of red blood cell velocity (VRBc) in a single capillary of rat before and after iontophoretic application of norepinephrine (NE, 3 mM, 30 set, current 70 nA). TFRis the time for 10% v,,, decrease from control, TRP, is the time for V,,, increase from the minimum V,,, toward the control level, and T,, is the time for reaching the control level. V,. and V,,, are the control and minimum V,,,‘s.

acid according to the fluorescence method of De la Torre (1980). For comparison with the previous rat mesentery preparation, the thin biceps femoris was mounted and stained to serve as the control preparation. Stained preparations were examined by means of a Leitz microscope (Metallux III) with a Ploemopak incident light fluorescence unit, a 50-W Mercury lamp, and filter set D. Experimental

Protocol and Data Analysis

The protocol consisted of two parts. The first part was designed to produce strong flow reductions, using NE concentrations similar to those reported in the rat mesentery. In rat TA muscles, 3 mM NE was applied to capillaries iontophoretically for 30 set using a current of 60-70 nA. In frog sartorius muscles, NE was applied both iontophoretically (3 mM, 30 set, 60-70 nA) and via microinjection (30 mA4, lo- to lOO-pl droplets). The second part of the protocol included weak iontophoretic stimulations of frog sartorius capillaries (3 PM, 1 set, 60-70 nA). The rationale was to use a stimulus that resulted in a barely detectable response. In both parts of the protocol, the majority of capillaries were stimulated only at one site chosen randomly along the capillary. It was ensured, however, that this site was at least 100-300 pm away from the feeding arteriole. We also excluded sites where an underlying arteriole was visible. In several experiments of both parts of the protocol, two stimulation sites of the same capillary were chosen. The first one was in the proximal portion of the capillary (100-300 pm away from the arteriole) while the second one was 500 pm downstream from the first site. In these experiments, equal amounts of NE were applied on both sites. Responses to stimulation were evaluated in terms of changes in the velocity of red cells, V,,, , measured either by the video flying spot technique (Tyml and Ellis, 1982) or by frame-by-frame analysis of video images. A stimulation was called successful when a decrease of V RBCwas more than 10% of the control velocity. In the first part of the protocol (strong stimulations), we also counted the number of capillaries with stopped flow crossing a test line oriented perpen-

FLOW

RESPONSE

-- b)

1200.- a)

77

TO NOREPINEPHRINE

t

4

0 “co m “MN

(n=42) (n43) /

RAT TlBlALlS ANTERIOR NE IONTOPHORESIS

3 mM

FROG SARTORIUS MUSCLE NE IONTOPHORESIS 3 mM

FROG SARTORIUS MUSCLE NE MICROINJECTION

30 mM

FIG. 2. Summary of V,,, responses in single capillaries to NE application in (a) rat tibialis anterior muscle using iontophoresis, (b) frog sartorius muscle (iontophoresis), and (c) frog sartorius muscle (microinjection). n is the number of stimulations in 10, 6, and 7 animals, respectively. *A significant difference between the control (V,,) and the minimum V,,, (VMIN) using t statistics (P 5 0.05).

dicularly to the muscle fibers. The line was positioned 250 pm downstream from the proximal site of NE application. In general, neither the TA muscle preparation nor the sartorius muscle preparation was suitable for arteriolar diameter measurements. Arterioles were poorly visible as they originated from the depth of the tissue. We observed, however, that at the very edge of the sartorius muscle (one or two muscle fibers thin) the last portion of the arteriole (up to 600 pm long) was sometimes visible enough to permit these measurements. We took advantage of this observation to obtain another measure of the NE response. Strong NE stimuli were applied iontophoretically on capillary sites 500-600 pm away from the feeding arteriole. Using a video caliper, diameters were measured directly from the video screen at sites with the clearest resolution of the arteriolar wall. Data (mean ? SD) were tested with either a paired or unpaired Student t test at P Z 0.05 (two tailed). RESULTS Strong Stimulations Single capillaries. Figure 1 shows an example of V,,, response to NE (3 mM in saline, applied for 30 set) in a single capillary of rat TA muscle. This response includes (i) a period of V,,, decrease, (ii) a period of low or stopped flow, and (iii) a period of reperfusion resulting in V,,, reaching the prestimulation level. In this figure the following parameters are defined, TFR (time for 10% V,,, decrease from control), TRp (time for V,,, increase toward control level, above 10% from the minimum VRBC), T,-, (t ime for reaching the control level), V,, (control V,,,), and If,,, (minimum V,,,). The parameter TFs (time for flow stop from the time of NE application) is not shown here. In general, responses among different capillaries varied considerably, both within the same species and within the iontophoresis and microinjection groups.

78

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AND

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Figure 2 summarizes results for “strong” stimulations in rat and frog muscles. Despite the large variability in data, it shows that all three types of stimulations resulted in significant decreases in V,,, , i.e., 80, 86, and 64%. Application of NE dissolved in distilled water (3 mM, 30 set, 70 nA) to 16 capillaries in three frogs resulted in a comparable V,,, decrease (75%). Table 1 shows detailed information regarding the reaction times TFR, TFs, TRp, and Tco, (NE in saline). It also shows the number of animals and capillaries used, and the actual number of stimulations. Values of TFR, TFs, and TRp were obtained from all stimulations. The value of T,, was obtained only from 75% of stimulations in which V,,, returned to a level that was either 90% or more of Vco. In the remaining 25% of stimulations, V,,, stabilized at a level between 50 and 90% of If,,. On average, the overall end velocity after recovery from the stimulations was 92%. In situations where multiple stimulations were carried out on one capillary, a minimum recovery period of 20-30 min was allowed between successivestimulations. Successrates for obtaining a V,,, reduction after the first-time stimulation of each capillary were 90, 100, and 85% for rat NE ionotophoresis, frog NE iontophoresis, and frog NE microinjection, respectively. In general, capillaries that did not respond the first time were not stimulated again. However, in the case of repeated stimulation with distal NE microinjection to capillaries in frog, the success rate increased from 73% (Table 2) to 91%. Control iontophoretic stimulations with saline and positive current, or with NE and negative current, showed no responses. Control microinjections with stained saline also had no effect on the blood flow. Applications of stain by iontophoresis and by microinjection showed patches of stain of 5 and 20-30 pm in diameter, respectively, that remained stationary around the tip of the micropipette. Table 2 shows reaction times for NE applied at the proximal site of the capillary and 500 pm downstream (distal site). Except for an increase in Tm from 15.7 to 24.2 set in the rat experiments, the proximal and distal reaction times did not differ statistically. Network responses. Responses to strong NE stimulations were not limited to the stimulated capillary. They were also present in neighboring capillaries fed by a common supplying arteriole. In general, the response was the strongest in about lo-12 capillaries branching from the terminal portion of the arteriole. The response in these capillaries appeared to start synchronously. However, flow stoppages did not necessarily occur at the same time. Flow in capillaries with the lowest control V,,, tended to stop first, while flow in capillaries with the highest VRBC last. The staggered stoppage times most likely reflected the variability in resistances to flow in individual capillaries. Capillaries in both species are known to vary appreciably in both their diameter and length (Safranyos et al., 1983; Plyley et al., 1976; Potter and Groom, 1983; Potter et al., 1989). Network responses showed that arterioles, rather than venules, were predominantly responsible for the response. Flow stoppages occurred only in capillaries supplied by the common arteriole. A constriction of the venule draining these capillaries could not occur, since those capillaries which drained into this venule but were supplied by a different arteriole never showed any stoppages. From our VR,, measurements in single capillaries it appeared that proximal applications caused stronger VR,, reductions than distal ones. However, because of the large variability of responses among capillaries, this tendency was not

FLOW

RESPONSE

TO NOREPINEPHRINE

79

Frog

Proximal Distal

30 30

3 3

6 7

5 6

10

9

N,

11 11

12 12

19

11

16 17

23 19

32

16

NC N,

TABLE 2

236.1 t 153.4 230.3 _s 163.7

408.5 -c 332.2 317.6 + 208.3

701.1 2 636.6

52.49 2 109.4 111.4§ k 183.6

60.76 k 136.0 43.83: t 83.4

355.9

19.46 +- 72.7

Vt”l,N (w/s4

205.61 t

..

Velocities

2 12.5

8.5

46.3 44.1

4 45.2 k 50.6

32.3 ? 37.3 28.63 k 16.6

24.2

15.7” i

TFR kc) 54.5

52.1 2 30.4 107.9 -t 86.1

64.4 k 46.9 78.1 t- 32.8

47.7 k 30.0

37.6 k

TFS (4

Reaction

115.5

143.4

640.4 2 380.3 765.2 t 618.0

343.5 2 262.4 349.2 2 177.7

157.8 t

173.6 t

TRP (W

times

166.6

1085.2 k 368.2 1101.9 2 806.7

392.6 k 228.8 548.9 + 487.3

27X.3 t

345.3 e 266.4

(s4

TCO

3 3

2 6

5

2

%a

7 5

IO 6

11

10

%s

91 73

100 100

84

100

(%I

Success rate

First-time stimulation

FOR PROXIMAL AND DISTAL APPLICATIONS OF NE IN SINGLE CAPILLARIES IN RAT AND FROG

778.7 2 403.0

(wlsec)

VCO

SD)

_---

Note. Symbols N,, N,, N, , TFR, TFs,TRp, T,,, nrR, and nFs are explained in Table 1. Vco, control Vnec; V,.,,, the minimum V,,, after NE application. PA significant difference between V,, and V,,, *A significant difference in TFRbetween proximal and distal application of NE in the rat.

(ionto.) Frog. (microinj.)

Proximal Distal

3

Distal

(ionto.)

3

Location

Proximal

Rat

NE (mM)

AND REACTION TIMES (t

Animal

VELOCITY

F

2

5

X

f3

g

FLOW

RESPONSE

81

TO NOREPINEPHRINE

0 PROXIMAL 633 DISTAL

* * Ik- !L (n=5)

NE MICROINJECTION

I

(n-10)

NE IONTOPHORESIS

3 mM 30 mM FIG. 3. Summary of network responses after NE application to single capillaries in the frog sartorius muscle. The response was measured in terms of capillary derecruitment, i.e., the number of capillaries with stopped flow. Derecruitment was significantly larger (p < 0.05) for proximal applications than for distal applications (i.e, 500 pm downstream from proximal site). The left two bars represent the maximum proximal and distal derecruitments measured 150 set after the microinjection of an NE droplet. The right two bars represent the maximum derecruitments measured 120 set following NE 30-set iontophoresis. n is the number of networks analyzed. Error bars represent SD. *Significant change at p < 0.05.

statistically significant. Network responses, on the other hand, showed this stronger response more consistently. In order to quantify the network response, we measured both the maximal capillary derecruitment and the derecruitment speed. Specifically, the maximal derecruitment was determined as the maximum number of visible capillaries with stopped flow crossing the test line drawn between the proximal and the distal sites of NE application. The maximal derecruitment could be measured only in frog experiments. Because of the larger size of red cells and capillaries in frogs, we could use a smaller magnification and a larger field of view (1.0 x 1.5 mm) where all visible derecruited capillaries could be counted. A smaller field of view in rats (0.4 x 0.6 mm) did not permit visualization of the maximal derecruitment. Instead, a derecruitment speed in rat (and also in frog to permit species comparison) was determined as the number of derecruited capillaries at 30 set after NE application. Figure 3 shows the network maximal derecruitment responses for frog iontophoresis and microinjection. In both stimulations distal applications showed a significantly weaker derecruitment. Figure 4 shows the capillary derecruitment speed. Proximal iontophoresis of NE in rat showed the strongest effect followed by frog iontophoresis and microinjection. Distal stimulations were weaker than the proximal ones. Arteriohr responses. In arterioles with measurable diameters, strong NE stimuli applied to capillaries resulted in vasoconstriction. We observed that within the visible portion of the arteriole, the proximal part of the arteriole (i.e, 200-600 pm upstream from the last bifurcation) constricted more than the distal part. Diameters were measured in this proximal part at sites with the best visibility. Among eight arterioles in five frogs, the control diameter was 28.5 -+ 6.7 pm. Constrictions could be seen as early as 20 set after the onset of NE stimulus. Measurements during the period of 60-120 set after this onset showed a significantly reduced diameter of 22.5 + 8.7. During the following period of about 10 min, diameters returned to the control level.

82

DIETRICH

AND

TYML

0

PROXIMAL

hzJ DISTAL

* hi(n=S)

FROG microinj. 30 mhi

FROG

RAT

ionto. 3mM

ionto. 3mM

FIG. 4. Summary’*of network responses in frog and rat measured in terms of the derecruitment speed. The speed was measured as the number of capillaries with stopped flow at 30 set after the end of NE application. It was significantly larger (P < 0.05) after proximal than after distal application in rat and frog iontophoretic experiments. The difference between proximal and distal derecruitment for microinjections in frogs was not significant at 30 sec. It became significant after 90 set and maximal at 150 set (Fig. 3). n is the number of networks analyzed. Error bars represent SD.

Weak Stimulations In our preliminary experiments we noticed that reducing both the NE concentrations and the application time resulted in an attenuated V,,, response. We used the 3-pM stimulus of 1 set duration to produce marginal V,,, reductions. Table 3 shows that among all applications in 10 capillaries in five frogs, the weak stimulus produced a small, but significant 9% V,,, decrease. When V,,, data were separated into proximal and distal applications, significant differences persisted between control and poststimulation VRBc’s. Table 3 also summarizes reaction times. There were no differences in reaction times between proximal and distal applications. Note that reperfusion times, TRPand Tc,, are much less than those for strong stimulations listed in Table 1. Histology Five TA muscles of five rats and three sartorius muscles of three frogs were stained with glyoxylic acid and examined for fluorescent adrenergic nerves. Stained longitudinal and cross sections of muscles showed nerve fibers around arterioles. The appearance of these fibers was similar to that demonstrated in the rat mesenter-y (Dietrich, 1989). However, in contrast to the mesentery, muscle sections did not show fibers near capillaries. We also stained the thin biceps femoris muscle in the rat to permit a comparison with the thin rat mesentery preparation. Similar to the sections in the TA muscle, nerves were present along arterioles in the biceps femoris, but not near capillaries. DISCUSSION Table 1 and Fig. 2 demonstrate that a local application of NE on a capillary in rat and frog skeletal muscle produced a temporary reduction of flow in the capillary. Figures 3 and 4 show that this reduction also occurred in a group of

Note.

Frog (ionto.)

(ionto.)

Frog

Animal

5 5

5

TABLE 3

VCO

VMIN (w/set)

49 25

238.7 2 141.2 271.5 k 93.5

between

24.4 t 16.7 19.0 + 8.1

22.8 2 14.8

TFR (-4

difference

212.68 _f 137.6 252.03: k 93.6

in Tables 1 and 2. §A significant

6 7

10 74 250.1 + 127.0 226.09 + 125.0

(w/set)

Velocities

k

31.1

V,,, and V,,,.

57.7 -t 31.1

57.7

TFS (set)

k

53.0 79.9 2 53.2 67.3 k 53.4

76.1

TKP 64

Reaction times

+

114.3 134.5 t 101.0 129.5 -c 142.6

132.9

TCO

(4

0 0

6

0

4

6

57

100

60

Success nFR nFs rate (%)

First-time stimulation

SD) FOR ALL WEAK NE STIMULI AND FOR STIMULI SEPARATEDINTO PROXIMAL AND DISTAL APPLICATIONS

Na NC N,

A11 symbols are explained

3 3

3

All applications

Proximal Distal

NE (WY

Location

VELOCITY AND REACTION TIMES (?

3

s

E

z

3 I2 z

E1

i! i+

E

2

?

84

DIETRICH

AND TYML

capillaries supplied by a common arteriole. Diameter measurements in arterioles of sufficient visibility in the frog muscle demonstrated a significant 21% constriction. To our knowledge, this phenomenon of flow reduction and constriction has been described previously only in the rat mesentery (Dietrich, 1989). In order to compare the present data with those of the mesentery, we used comparable millimolar concentrations of NE. Micromolar concentrations were used to produce just detectable responses. Referring to Table 1, reaction times found in the rat muscle were similar to those of the rat mesentery. Also similar to mesentery, weak stimulations in the frog muscle resulted in less frequent and shorter-lasting responses than those of strong stimulations (Tables 1 and 3). The present millimolar concentrations were much higher than NE levels in blood needed to produce vascular constrictions (Handa and Duckles, 1987). It should be noted, however, that NE concentrations near capillaries might have been much less. Curtis et al. (1960) demonstrated that the concentration of material ejected iontophoretically decreases rapidly with distance. In our experiments, NE needed to cross the connective tissue at the muscle surface and a 20- to 40-pm distance through muscle tissue before reaching a capillary. The use of millimolar concentrations was also consistent with the literature. Typically, up to molar concentrations of materials were used for recent iontophoretic work (Segal and Duling, 1989; Stone, 1985). Our study also showed that the use of saline and distilled water as NE solvents produced comparable V,,, reductions. The capillary bed visible at the surface of both rat tibialis anterior and frog sartorius muscles is supplied by arterioles rising vertically through the depth of the tissue. This feature was advantageous since NE could be applied on capillary sites away from any arterioles. However, because of the tissue depth, arterioles were difficult to see in sharp focus and therefore their diameters were difficult to measure. Although we did succeed in obtaining these measurements at special sites in the sartorius muscle, the VRBCproved to be a more practical and a more sensitive measure of the NE response. A key requirement for using this measure was the stability of V,, both before NE application and after the expected time of V aBCreturn to the control level (6-7 min for iontophoresis, 17 min for microinjection). We often observed that either spontaneous (Tyml and Groom, 1980) or experimentally induced fluctuations of V,,, (Tyml et al., 1990) could mask the NE response. We also observed that, in the frog, a long-lasting dilatory stimulus associated with a high V,,, (Tyml, 1986) prevented a large reduction of V RBCupon NE stimulation. Similarly, a very low control VRBC (

Microvascular flow response to localized application of norepinephrine on capillaries in rat and frog skeletal muscle.

Recently, Dietrich (1989, Microvasc. Res. 38, 125-135) demonstrated that a local application of a minute amount of norepinephrine (NE, 5.5 mM, 0.01-88...
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