Peptides,Vol. 13, pp. 1181-1185, 1992

0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.

Printed in the USA.

Microvascular Effects of Atrial Natriuretic Peptide in Rat Cremaster R. W A Y N E BARBEE, LISA M. H A R R I S O N - B E R N A R D A N D P A M E L A K. C A R M I N E S l

Division of Research, Alton Ochsner Medical Foundation, New Orleans, LA 70121 and Department of Physiology, Tulane University School of Medicine, New Orleans, LA 70112 Received 6 M a r c h 1992 BARBEE, R. W., L. M. HARRISON-BERNARD AND P. K. CARMINES. Microvasculareffects of atrial natriureticpeptide in rat cremaster. PEPTIDES 13(6) 1181-1185, 1992.--Experiments utilized the open cremaster preparation to test the hypothesis that atrial natriuretic peptide (ANP)-induced volume changes result from microvascular resistance alterations. Atrial natriuretic peptide (25, 100, and 500 ng/kg/min, IV) or vehicle was infused into anesthetized rats. At the two highest ANP infusion rates, mean arterial pressure was significantlyreduced from 104 + 3 (control) to 87 _+2 and 77 + 2 mmHg, respectively. Hematocrit was 41.0 _ 0.8 and 45.6 + 0.9% (p < 0.05) at the end of vehicle and ANP infusions, respectively. Despite these effects of ANP, there were no significantarteriolar or venular diameter alterations. Thirty uM nitroprussidesignificantlydilated all vesselsegments except large venules. These observations suggest that resistance alterations in the skeletal muscle microvasculature are not the cause of ANP-induced fluid movement. Microcirculation Skeletalmuscle Vascular smooth muscle

Hematocrit

Central venous pressure

DESPITE more than a decade of intensive research since the original discovery of atrial natriuretic peptide (ANP), many questions remain unanswered regarding the hypotensive properties of this peptide. The ability of ANP to lower mean arterial blood pressure was originally believed to be the result of a reduction in total peripheral resistance, since both atrial extracts and synthetic peptides had been shown to relax preconstricted vascular smooth muscle (31). However, data obtained from in vivo experiments do not support this prediction. Although bolus administration of this peptide generally causes a transient decrease in vascular resistance (14,28), acute infusions normally lower arterial pressure by decreasing plasma volume (27), leading to a fall in cardiac output (13). The decrease in plasma volume occurs even in nephrectomized rats, suggesting a shift of fluid from the intravascular to the interstitial space (27). This loss of fluid occurs in several tissues at pathophysiological and pharmacological infusion rates of ANP (29,30,32). The fluid extravasation induced by ANP could be caused by a number of mechanisms, including an increase in capillary permobility (19), capillary surface area (22), or capillary hydrostatic pressure. The hydrostatic pressure effect might result from arteriolar dilation and/or venular constriction. A role for increased venous resistance has been suggested, based on whole-animal studies (5,15); howe,Jer, these results have not yet been corroborated by observations at the microcirculatory level. A large portion of the whole-animal transcapillary fluid shift induced by ANP occurs in skeletal muscle. Atrial natriuretic peptide infusion rates of 50-500 ng/kg/min cause labeled al-

Arterioles

Venules

bumin accumulation (a marker of transcapillary fluid shifts) in skeletal muscle (32); therefore, the skeletal muscle microcirculation should be useful for investigating mechanisms of ANP's hypotensive action. We, therefore, utilized the open cremaster preparation (10) to test the hypothesis that ANP exerts its effects on skeletal muscle through alterations in microvascular resistances at precapillary and/or postcapillary sites. METHOD

Surgical Procedures Experiments were performed in 37 male Sprague-Dawley rats (183 _+ 2 g body wt.) provided normal chow and water ad lib. Rats were anesthetized with Inactin (100 mg/kg body wt. IP) and placed on a temperature-controller pad to maintain rectal temperature at 37 °C. Following tracheostomy, the right carotid artery was cannulated for sampling of hematocrit (Hct) and monitoring systemic arterial pressure using a Statham P23XL transducer and polygraph (Grass Instruments, Quincy, MA). A cardiotachometer, triggered by the pulsatile arterial pressure signal, was utilized to determine heart rate (HR). The right jugular vein was cannulated to allow intravenous infusions. The left femoral or jugular vein was cannulated, and the tip of the cannula advanced to the right atrium, for measurement of central venous pressure (CVP). The right cremaster muscle was exposed for microvascular study according to the method described by Faber et al. (10). The muscle with intact innervation and circulation was dissected

t Requests for reprints should be addressed to Pamela K. Carmines, Ph.D., Department of Physiology #SL39, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112-2699. 1181

1182 free of the testicle and suspended as a fiat sheet over an optical port in a bath of 40 ml. The bath was filled with modified Kreb's solution consisting of I 13 mMNaCI, 25 mMNaHCO3, 4.7 m M KCI, 2.6 m M CaC12.2H20, 2.4 m M MgSO4, 1.2 m M KH2PO4, and 11.6 m M dextrose. Both the tissue bath and the Kreb's stock reservoir were maintained at 35°C and bubbled with N2 and CO2 to maintain Po2 (20-30 mmHg), Pco2 (35-45 mmHg), and pH (7.35-7.45). When muscle twitching occurred, this activity was minimized by addition ofpancuronium bromide (0.8 ug/ml) to the bath. This treatment, which was necessary in only a few experiments, has been shown not to alter vessel diameter (10). The preparation was positioned on the fixed stage o f a Nikon Optiphot trinocular microscope. First-order vessels were observed using the × 10 objective (numerical aperture = 0.22), while second-order vessels were examined using a ×20 long-distance objective (numerical aperture = 0.25). A water immersion ×40 objective (numerical aperture = 0.75) was used to inspect thirdand fourth-order vessels. A Newvicon camera (Dage-MTI, Michigan City, IN) was used to provide video images of the microvessels. These images were processed by an image enhancer (MFJ Enterprises, Starkville, MS) and displayed at ×250-1000 on a high resolution monitor (Conrac Display Systems, Covina, CA). Images were recorded by an S-VHS format videocassette recorder (Panasonic, Secaucus, N J) for later playback and analysis. Microvessel inside diameters were measured from videotaped images using a calibrated digital image shearing monitor (Instrumentation for Physiology & Medicine, San Diego, CA). This system allows vessel diameter measurements reproducible to within 1 #m. Microvessels were classified according to their branching pattern, with the largest central arteriole entering the muscle designated as the first-order arteriole ( 1A) and its attendant venule designated l V. Branches from the I A were designated secondorder arterioles (2A), which were associated with second-order venules (2V). Subsequent branches of the vasculature were numbered consecutively. Previous studies have documented similar behavior of first- vs. second-order vessels and third- vs. fourth-order vessels in response to various humoral and physical stimuli (8,10). Therefore, results from first- and second-order vessels were combined, as were data from third- and fourthorder vessels. The acceptability of the cremaster preparation was judged based on the following criteria: 1. 2. 3. 4. 5.

no gross signs of tissue trauma, no leukocyte adhesion to venular walls, vasomotion evident in third- and fourth-order arterioles, rapid blood flow through small arterioles, and capillary flow showing intermittency.

Vessels were selected for study based on clear visibility of inside diameter. In addition, only 3A and 4A vessels that could be shown to eventually give rise to capillaries were selected for study.

Experimental Protocols Upon the completion of surgery (approx. 75 min), the animals were allowed at least 30 rain recovery. Each rat was randomly assigned to one of the two treatment regimens: ANP infusion or vehicle infusion. Rats in the ANP group received vehicle (0.9% NaC1, l0 ~d/min IV) during a 30-min control period. Images of the microscope field containing the one or two vessels selected for study were videotaped in 2-min segments at the beginning, midpoint, and end of the control period. Then the vehicle infusion was halted and an infusion of synthetic rat ANP [ANP(99126), Peninsula Laboratories, Belmont, CA] was begun at the same rate. Atrial natriuretic peptide was administered to each

BARBEE, HARRISON-BERNARD AND CARMINES rat at increasing doses (25, 100, and 500 ng/kg/min, 45 min each dose). These infusion rates have been shown to result in physiological (490 + 110 pg/ml), pathophysiological (1232 + 199 pg/ml), and pharmacological (7734 +_ 675 pg/ml) plasma levels of ANP, respectively (3,32). Images of the microvessel(s) under study were videotaped at 10-min intervals (2-min duration) throughout each ANP infusion period. During the last 10 rain of each infusion period, approximately 75 /zl of arterial blood was withdrawn in duplicate for measurement of Hct. At the end of each experiment, sodium nitroprusside (NP) was added to the cremaster bath (final concentration = 30 uM) to document the vasodilatory responsiveness of the microvasculature. Rats in the vehicle infusion group were treated identically, except that ANP was not included in any intravenous infusion. Vessel diameters were determined from videotape at a single measurement site at 10-s intervals. Measurements obtained during the final 30 min of each infusion period were averaged to provide an indication of the plateau response to each experimental manipulation. A separate group of identically prepared rats (n = 5) was utilized to document microvascular responsiveness to vasoconstrictor stimuli in our cremaster preparation. A single third-order arteriole (3A) was chosen for study in each rat. After a 30-min control period, norepinephrine (NE) was added to the bath to achieve final concentrations of 3, 30, 300, and 3000 n M (10 min each concentration). Vessel diameters were measured at 30-s intervals.

Data Analysis Vessel diameters and hemodynamic parameters were statistically analyzed using two-way analysis of variance (ANOVA) for repeated measures with a post hoc Tukey's test to locate significant differences within and between appropriate groups. A t-test was used to compare the mean change in diameter to NP when compared to the previous period for a given vessel classification. Values for p less than 0.05 were considered significant. All data are reported as means _+ SEM. RESULTS

Effects of ANP on Systemic Hemodynamic and Fluid Variables The effects of ANP infusion on systemic hemodynamics and Hct are illustrated in Fig. 1. During the control period, MAP averaged 104 +__ 3 (n = 20) and 1 0 7 _ 4 ( n = 17) m m H g i n vehicle and ANP groups, respectively. Animals in the vehicle group exhibited a slow progressive decline in MAP, achieving statistical significance in the final experimental period. Low-dose ANP infusion had no significant effect on blood pressure; however, the intermediate and high-dose infusion of ANP lowered MAP by 17 and 27 mmHg, respectively. The reduction in MAP elicited by intermediate and high-dose ANP infusions differed significantly from that observed in rats receiving vehicle alone. Despite the hypotensive effect of ANP, HR was significantly lower during high-dose ANP infusion compared to vehicle-infused animals (398 --- 14 vs. 423 ___13 bpm). The pharmacological dose of ANP also reduced CVP significantly ( - 0 . 4 ___0.1 mmHg, n = 15, p < 0.05) compared to both the control period and vehicle-infused animals. Moreover, Her was elevated during highdose ANP infusion (44.3 ___0.7%, n = 10) compared to vehicle infusion (41.5 +__0.8%, n = 15). Therefore, both pathophysiological and pharmacological doses of ANP had significant hemodynamic effects, similar to those previously observed (27).

ANP AND SKELETAL MUSCLE MICROCIRCULATION

120

bath following infusion of A N P or vehicle. This treatment caused a significant dilation of all vessels examined except large venules (Fig. 3). Vasoconstrictor responsiveness of the cremaster preparation was assessed in a separate group of rats. Third-order arteriolar diameter in these rats averaged 26 ___ 2 u m (n = 5) under control conditions, a value not significantly different from 3A diameters in the ANP- and vehicle-infused rats. Sequential addition of norepinephrine to the bath (3-3000 nM; Fig. 4) elicited dose-related reductions in 3A diameter, with significant vasoconstriction observed at norepinephrine concentrations of 30 n M and greater. The highest concentration of N E caused vessel constriction to 1.8 + 0.7 urn.

110

A

E E v ,,,¢ "5

100 90

80

*t

70 60

I

I

I

I

475

I

I

I

I

450 E

CL .JD

425

p,. "r"

400

DISCUSSION

375

-r

v

E E

Q. > 0

1183

t

350

I

I

I

I

0.4

I

i

i

i

I

I

I

These results indicate that intravenously infused A N P has no influence on innervated cremaster muscle microvascular diameters at doses that clearly affect MAP, CVP, and Hct. Although not measured in this study, we have previously shown that high-dose A N P infusion (500 ng/kg/min) decreases cardiac output by 24-29% (2). The hypotensive effect of A N P in vivo

0.2 0.0 -0.2

-0.4 ,t

-0.6

-0.8

120

I

110 100

4B

90

46 ~"

44

"~ -1.

42

80

40 38

I

I

I

I

70

I

I

I

I

120

i

i

i

i

70

I

I

0

25

'j I

I

100

500

t

120 110 100

ANP DOSE (ng/kg/min)

FIG. 1. Systemic hemodynamic responses to vehicle (open circles, 0.9% sodium chloride, 10 #l/min) or graded infusions of ANP (closed circles, 25, 100, and 500 ng/kg/min, 45 min per dose). MAP, mean arterial pressure; HR, heart rate; ACVP, change in central venous pressure; Hct; hematocrit. *p < 0.05 compared to zero dose ANP. ?p < 0.05 compared to vehicle infusion.

o

90

8

80

tJ

LIJ I-W

Microcirculatory Effects The effects of intravenous A N P infusion at various doses on diameters of cremaster muscle venules and arterioles are illustrated in Fig. 2. There were no significant differences between resting (control) diameters of vehicle- vs. ANP-infused rats. Under control conditions, diameters o f large (1A, 2A) and small (3A, 4A) arterioles averaged 84 + 10 # m (n = 12) and 18 + 2 p m (n = 12), respectively. Diameters of large (IV, 2V) and small (3V, 4V) venules were 127 + 12 g m (n = 12) and 33 ___ 3 # m (n = 11), respectively. There were no statistically significant changes in luminal diameter of any microvascular segment in the vehicle-infused rats. Diameters of large arterioles tended to decrease during A N P infusion; however, these changes were smaller than those observed in vehicle-infused animals and were not statistically significant. Small arterioles showed no change in diameter with A N P infusion. Large venules showed no tendency toward dilation or constriction with A N P infusion. Small venules exhibited a slight vasodilatory tendency during A N P infusion (105 ___5% of control at high-dose ANP); however, this effect was not statistically significant. To document the vasodilatory ability of the cremaster microvasculature in these animals, N P (30 gM) was added to the

"5