Delayed Arteriolar Relaxation After Prolonged Agonist Exposure: Functional Remodeling Involving...

American Journal of Physiology H00986-2002R2 Final Accepted Version

Delayed Arteriolar Relaxation After Prolonged Agonist Exposure: Functional

Remodeling Involving Tyrosine Phosphorylation.

Michael A. Hill1, Simon J. Potocnik1, Luis A. Martinez-Lemus2,

and Gerald A. Meininger2

1Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora,

Victoria 3083, Australia and 2Department of Medical Physiology, College of Medicine,

Texas A&M University, College Station, Texas 77843, USA.

Running Title: Functional Remodeling in Arterioles

Address for Correspondence:

Michael A. Hill, PhD.

Microvascular Biology Group

School of Medical Science

RMIT University

Bundoora, Victoria 3083

AUSTRALIA

Ph: 61-3-9925-7754

FAX: 61-3-9925-7628

e-mail: [email protected]

Copyright (c) 2003 by the American Physiological Society.

Articles in PresS. Am J Physiol Heart Circ Physiol (April 24, 2003). 10.1152/ajpheart.00986.2002

American Journal of Physiology H00986-2002R1 24/04/2003

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Summary:

While arteriolar contraction is dependent on Ca2+- induced myosin phosphorylation other

mechanisms including Ca2+ sensitization and time-dependent phenomena such as cytoskeletal

and cellular reorganization may contribute to contractile events. We hypothesized that if

arteriolar smooth muscle exhibits time-dependent behavior that this may be manifested in

differences in relaxation following short and long-term exposure to contractile agonists.

Studies were conducted in isolated arterioles, pressurized to 70 mmHg. In initial experiments

(n = 10), rate of relaxation was measured following acute (5 min) or prolonged (4 hr)

exposure to 5µM norepinephrine (NE). Prolonged exposure to NE resulted in a significantly

(P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of

vessels exposed to NE for 4 hrs was observed following superfusion with 0 mM Ca2+ buffer

indicating that the alteration in relaxation was reversible and Ca2+ dependent. A similarly

impaired dilation was not observed with 4 hr exposure to KCl (75 mM). To determine

mechanisms contributing to the effects of prolonged NE exposure studies were performed in

the presence of the microtubule depolymerizing agent demecolcine (10µM) or a series of

tyrosine phosphorylation inhibitors. While demecolcine caused significant vasoconstriction

(P < 0.05) and potentiated NE vasoconstriction it did not prevent the effect of long-term NE

exposure on relaxation. Genistein, while having no effect on acute NE-induced contraction,

concentration-dependently inhibited prolonged NE constriction. Similarly, the Src (PP1) and

p42/44 MAP kinase (PD98059) inhibitors prevented maintenance of long-term NE

contraction. The data indicate that prolonged exposure to NE induces biochemical alterations

that impair relaxation following removal of the agonist. The contractile effects are Ca2+

dependent, involve tyrosine phosphorylation, but do not appear to involve the polymerization

state of the microtubule network.


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Key Words: arterioles; remodeling; myogenic; agonist; Ca2+; cytoskeleton, tyrosine

phosphorylation.


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Introduction

Contraction of arterioles in response to both agonists and increased intravascular

pressure has been shown to be largely dependent on the classical smooth muscle biochemical

pathway involving Ca2+-calmodulin induced myosin regulatory light chain phosphorylation

(for reviews see references 10, 16, 18 and 36). The requirement for these pathways has been

directly confirmed in studies of isolated arterioles in combination with fluorescence

measurements of intracellular Ca2+ and electrophoretic estimations of protein phosphorylation

(26, 33, 48, 49). However, it is now appreciated that other mechanisms including Ca2+

sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization

may contribute to contractile responsiveness (7, 15, 30, 38, 40, 41). These mechanisms may

support the Ca2+-myosin light chain phosphorylation pathway and, further, provide a

mechanism for maintained tension development at comparatively low energy cost. The latter

suggestion appears intuitively relevant to arterioles given that these vessels typically

demonstrate a maintained state of partial contraction or tone – a phenomenon important for

the establishment of vascular resistance and necessary for the function of vasodilators.

Historically, many studies examining pathways activated by contractile agonists have

been limited to relatively acute exposures to these agents. Whereas, studies aimed at

delineating events related to compensatory growth responses, such as remodeling in

hypertension, have often considered long-term responses to trophic stimuli including

catecholamines and angiotensin II (4, 5). Further, growth-related studies have been often

performed on cultured cells which typically exhibit phenotypic changes compared to cells in

the in vivo state. It is likely, however, that rather than distinct mechanisms there exists a

continuum of responses influenced by both the duration of exposure to the contractile

stimulus and possibly the contractile response itself. It has, therefore, been suggested that the

functional properties of smooth muscle are themselves altered by events such as


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reorganization of the cytoskeleton as the tissue undergoes adaptation to environmental factors

(15). As an extension of this we hypothesized that vascular remodeling may begin at a much

earlier time frame and be initially characterized by alterations in cytoskeletal properties

and/or the physical relationship between adjacent smooth muscle cells. This latter alteration

being considered a property of cell-cell and cell-matrix interactions. Support for the

occurrence of such rearrangements is provided by studies of cultured vascular cells exposed

to agonist and mechanical stimuli (8, 34).

With respect to the above general hypothesis it would be expected that if arteriolar

smooth muscle exhibits time frame-dependent changes in cytoskeletal or cellular arrangement

that this may be manifested in differences in the rate and extent of relaxation when

comparing vascular responses to short and long term exposure to a contractile agonist.

Therefore the aim of the present studies was to compare acute (5 min) and prolonged (4 hr)

exposures to contractile stimuli and to examine the involvement of candidate signaling

pathways.


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Materials and Methods:

Animal:

The studies used 59 male Sprague-Dawley rats weighing between 200 and 350g.

Before experiments, rats were housed in pairs in a dedicated animal facility with a 12:12 hr

light-dark cycle. During this period rats were allowed free access to a standard rat chow and

drinking water. All procedures were approved by the Animal Ethics and Experimentation

Committee at RMIT University.

Isolated Arteriole Preparation :

Rats were anesthetized with sodium pentothal (100mg/kg intraperitoneal) after which

the right and/or left cremaster muscle was exteriorized, excised from the animal and placed in

a cooled (4oC) chamber containing dissection buffer (3mM 3-N-morpholino propanesulfonic

acid (MOPS); 145mM NaCl; 5mM KCl; 2.5 mM CaCl2; 1mM MgSO4; 1mM NaH2PO4;

0.02 mM EDTA; 2mM pyruvate; 5mM glucose and 1% albumin) (12). Segments (1.2 – 2.2

mm in length) of the main intramuscular arteriole (1A) were dissected from the muscle as

previously described (26, 48). Vessel segments were then cannulated with glass

micropipettes, secured using 10-0 monofilament suture and mounted in a custom-designed

tissue chamber (volume 5 ml). The vessel preparations were then positioned on the stage of

an inverted microscope. The cannulated arterioles were continually superfused (0.5 - 4

ml/min.) with a physiologic buffer solution, containing, 111mM NaCl, 25.7mM NaHCO3,

4.9mM KCl, 2.5mM CaCl2, 1.2mM MgSO4, 1.2mM KH2PO4, 11.5mM glucose and 10mM 2-

N-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). Vessel segments were

gradually pressurized to 70 mmHg and warmed to 34oC during a 60 minute equilibration

period. To be suitable for use in studies vessels had to be free of pressure leaks and develop


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spontaneous basal tone as evidenced by an active diameter between 40 and 65% of passive

diameter at 70 mmHg.

Measurement of vessel diameter (in µm) was performed on-line using an electronic

video caliper. Changes in vessel diameter following exposure to a given treatment were

typically normalized to passive (maximally dilated with 0 mM Ca2+ buffer containing 2 mM

EGTA) diameter at 70 mmHg to account for slight variation in absolute vessel diameters

observed between preparations.

In experiments requiring measurement of changes in intracellular Ca2+, vessels were

incubated (60 min, room temperature) with 2µM fura 2-acetoxy methylester (Molecular

Probes, Eugene, OR) in buffer containing 0.5% DMSO and 0.01% pluronic. The abluminal

surface of the vessel segment was exposed to the fura 2-AM solution to restrict dye loading

to the vascular smooth muscle layer (Meininger et al., 1991). The dye loading procedure was

followed by a 30 min washout period with the physiologic salt solution. Fura 2 loaded

vessels were exposed to epi-illumination (75W Xenon source) with light of alternating

excitation wavelengths (340 and 380 nm) using a motor-controlled filter wheel (20 Hz).

Fluorescence emission was transferred from the microscope to a photomultiplier tube

(Hamematsu, Bridgewater, NJ) coupled to a signal processor. (Texas A&M University,

College Station, TX). Simultaneous trans-illumination with wavelengths greater than 610 nm

provided a non-fluorescent image that enabled measurement of internal arteriolar diameter

while fluorescence data were collected. This procedure did not interfere with measurements

of Ca2+-related fluorescence. Fluorescence emission intensities were expressed as the

340/380 nm ratio to allow quantitative estimates of changes in arteriolar wall intracellular

Ca2+. Details of theses procedures have been presented in previous publications (17, 26, 48).


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Experimental Protocols:

Acute and Prolonged Exposure to Norepinephrine

After establishing baseline diameter, arterioles were exposed to norepinephrine (NE,

5µM) until a maximal contraction was achieved (approximately 5 min). The superfusate was

then rapidly changed and vessel diameter and time recorded as the NE was washed out.

Following return to basal diameter, or after 15 minutes superfusion with fresh physiological

salt solution (chosen as a predetermined surrogate endpoint), vessels were subsequently

exposed to adenosine (3 x 10-4M) and then 0 mM Ca2+ buffer containing 2 mM EGTA to

obtain a measurement of maximal passive diameter. Vessels were then superfused with Ca2+

containing buffer for 20 min to re-establish basal conditions. The exposure to NE was then

repeated with the exception that the contraction was maintained for 4 hr before washout as

described above. Dilator responses to adenosine and 0 mM Ca2+/2mM EGTA buffer were

similarly repeated.

Time control studies were conducted to determine whether vessels responded

similarly to acute NE exposures performed 4 hrs apart. A further set of control experiments

were conducted to determine whether the initial acute exposure to NE exerted a

preconditioning effect. In these experiments, relaxation responses to adenosine (10µM) were

compared prior to exposure to NE (that is in the presence of spontaneous myogenic tone

alone at 70 mmHg intraluminal pressure) and to a similar adenosine exposure following 4hrs

of NE (5µM) exposure. NE was washed out, as for the above experiments, prior to addition

of adenosine.

In a separate set of vessels (n = 7) loaded with Fura 2 (2 µM) the above protocol was

repeated to examine Ca2+ responses during the acute and prolonged NE exposures.

Experiments were performed in both the presence and absence of Fura 2 loading to determine

any possible buffering effects due to dye loading.


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Short and Long-term Exposure to KCl

To examine the effect of contraction per se the above studies were repeated in a

separate set of arterioles (n = 5) substituting KCl (75 mM) as the contractile agent. Short

and prolonged exposures to KCl were performed as described above.

Effect of Microtubule Depolymerization on Arteriolar Responses to Acute and Prolonged

Exposure to Norepinephrine

Previous studies in our laboratories had shown modulation of arteriolar

contractile responses to NE by the polymerization state of the microtubular network (37).

Therefore to examine the possible involvement of the microtubular network in the differences

in contractile responses to acute and prolonged NE exposure, experiments were performed in

the absence and presence of the depolymerizing agent demecolcine (10 µM). The response

of arterioles to acute NE (5 µM) exposure was studied in the absence of the agent after which

vessels were exposed to demecolcine for 60 min and the acute NE response repeated. The

prolonged response (4 hr) to NE was then determined in the continued presence of

demecolcine. Conditions for these experiments were established in previous studies (37, 38).

Effect of Tyrosine Phosphorylation Inhibition on Arteriolar Responses to Acute and

Prolonged Exposure to Norepinephrine

To examine whether tyrosine phosphorylation-dependent events contribute to the

differences in contractile responses to acute and prolonged NE exposure, experiments were

performed in the absence and presence of genistein (3, 10 and 30 µM). The response of

arterioles to acute NE (5 µM) exposure was studied in the absence of the inhibitor after which

vessels were exposed to genistein for 20 min and the acute NE response repeated. The


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prolonged response (4 hr) to NE was then determined in the continued presence of the

tyrosine kinase inhibitor. Only one concentration of genistein was examined in any given

arteriolar segment.

In addition to the studies of the non-specific tyrosine kinase inhibitor (genistein)

studies were conducted in the presence of the Src inhibitor PP1 (10 µM) and the MEK

inhibitor PD98059 (50 µM) to prevent activation of P42/44 MAP kinase. Studies were

performed according to the protocol used for genistein. The rationale for examining the

effects of these inhibitors was based on studies implicating the involvement of Src and MAP

kinase activation in both agonist and pressure-induced smooth muscle responses (24, 25, 46).

Drugs and Chemicals

Buffer salts and chemicals were obtained from Sigma unless otherwise stated. NE

(arterenol) was dissolved in 1mM ascorbic acid as a stock solution at 10-2M and diluted in

krebs solution to 5 µM in 100 ml quantities as required through out the protocol. Working

solutions of NE were protected from light and all solutions were kept below room

temperature (either on ice or on a ‘cold’ block). A stock solution of adenosine was prepared

in Krebs at a concentration of 10-2 M and added directly to the arteriole bath (5 ml) to achieve

a concentration of 3 x 10-4M. PD98059 (Biomol Research Laboratories, Plymouth Meeting,

PA, USA) dissolved in DMSO at a concentration of 10-1 M was stored at –20oC in 50 µl

aliquots and diluted to 50 µM in krebs containing DMSO (600µl/100ml) for each experiment.

Frozen 100 µl aliquots of PP1 (Biomol Research Laboratories, Plymouth Meeting, PA, USA)

in DMSO at a concentration of 10-2M were diluted in krebs to 10 µM for each experiment.

Demecolcine dissolved in DMSO (1 mM) was further diluted in Krebs to 10 µM.

Concentrations of norepinephrine, adenosine, genistein, PD98059, PP1 and

demecolcine used were based on previous studies (26,30, 32). In addition, PD98059 was


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shown to inhibit phosphorylation of p42/44 MAP kinase in response to epidermal growth

factor or increases in intraluminal pressure as determined by gel electrophoresis/western

transfer (data not shown).

Statistics and Data Handling

To account for differences in starting vessel diameters data has been normalized

according to the following equation: Diameter (% max) = (diameter under a given

experimental condition/diameter in 0mM Ca2+ buffer containing 2mM EGTA) X 100. All

measurements were taken at an intraluminal pressure of 70 mmHg and maximal diameters

were measured prior to prolonged NE exposure.

Differences in group data, for the effect of a given treatment, were determined using

analysis of variance (ANOVA). When the ANOVA indicated an overall significant change,

differences between individual groups were determined using the t-test. Results are

presented as the mean ± standard error of the mean (SEM) and differences were considered

significant when P < 0.05, n represents both the number of arteriole segments and rats

studied.


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Results:

Arterioles used in these studies had active diameters of 80.8 ± 2.4 µm at 70 mmHg in

the absence of agonists and/or inhibitors. Passive diameters, obtained in 0mM Ca2+/2mM

EGTA prior to agonist/inhibitor protocols, were 153.8 ± 1.8 µm. Diameter measurements are

subsequently presented as a percentage of this passive diameter and are termed % maximal

diameter.

Acute and Prolonged Exposure to Norepinephrine

Acute exposure to NE (5 µM) resulted in a rapid contraction from 46.9 ± 3.9% to 23.9

± 1.7% of maximal diameter which was subsequently maintained during the prolonged

exposure (26.9 ± 1.7 % of maximal diameter at 4 hrs; n.s. compared to acute exposure)

(Figures 1 and 2). Despite reaching a similar level of vasoconstriction, subsequent washout

of the mechanical response with fresh buffer was significantly (P < 0.05) delayed in arterioles

subjected to the prolonged NE exposure. In 10 arterioles acutely exposed to NE, baseline

diameter was reached in 2:17 ± 0:45 minutes after initiating washout with fresh buffer. In

contrast, 7 of the 10 vessels remained constricted for periods of greater than 15 minutes when

washout followed the prolonged (4 hr) NE exposure. After 4hr exposure to NE, arterioles

also exhibited an impaired relaxation response to topically applied adenosine (3 x 10-4M)

although full relaxation was attained on removal of extracellular Ca2+ (Figures 1 and 4).

Control experiments comparing acute responses to NE at time 0 and 4 hrs later

indicated that the delayed relaxation following the prolonged NE exposure was not due to

time-dependent changes in the preparation independent of the action of the α-agonist.

Similarly, an additional series of vessels (n = 6) were studied to examine the effect of 4 hr NE

exposure in the absence of an acute pre-exposure to the agonist to exclude any effect

resulting from pre-conditioning. Consistent with impaired relaxation following prolonged

exposure to NE, after 4 hrs superfusion with 5 µM NE vessels dilated to 78.3 ± 3.7 % of


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maximal diameter in response to adenosine (10-4M) compared to 92.2 ± 0.9 % prior to NE

exposure (p < 0.05). As above, arterioles exposed to NE for 4 hrs dilated to maximum when

superfused with 0 mM Ca2+/2mM EGTA buffer.

In contrast to NE, exposure to KCl (75 mM) did not impair subsequent relaxation

responses (Figure 2). Further, unlike the NE-induced responses, KCl contractions tended to

wane over the 4hr time period (Figure 2).

Vessels loaded with the Ca2+-sensitive dye Fura 2 showed a similar impairment in

relaxation following prolonged NE exposure. NE (5µM) caused a transient increase in Ca2+

that declined towards baseline over the period of prolonged agonist exposure despite the

maintained vasoconstriction. Comparison with time controls, not exposed to NE,

demonstrated that after 30 – 60 min exposure to the α-agonist that Ca2+i levels were not

statistically different from baseline (Figure 3).

The Ca2+ response did not appear to be a function of pre-exposure to NE or time as

peak changes were 178.9 ± 7.3 % (relative to values in 0 Ca2+ buffer) during the first NE

treatment compared to 183.0 ± 10.9 % in response to the second NE exposure (not

significantly different). Similarly, after 5 mins exposure to NE Ca2+ had plateaued to 139.4 ±

4.2 % and 134.2 ± 6.3 % (not significantly different) respectively.

Effect of Microtubular Depolymerization

One hour demecolcine (10 µM) treatment caused a decrease in baseline diameter and

potentiated NE-induced constriction as seen in our previous studies (36, 37) (see Table 1).

Despite the presence of the microtubule depolymerizing agent, arterioles maintained the

prolonged NE constrictor response and continued to show the impaired relaxation on wash

out of the agonist (Figure 5; Table 1).


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Effect of Tyrosine Kinase Inhibitors

The initial series of studies examined the effect of the general tyrosine kinase

inhibitor, genistein, at 3, 10 and 30 µM. Genistein caused a concentration-dependent loss of

basal tone but did not significantly inhibit the acute contractile response to NE (5µM) (Table

1). In contrast, genistein treatment resulted in a concentration-dependent loss of the NE-

induced constriction over time despite the continued presence of the agonist (Figure 6; Table

1).

The Src inhibitor, PP1 (10 µM, n = 4), did not affect either the basal level of

myogenic tone nor the acute contraction to NE (Figure 6b; Table 1). Similarly to genistein,

however, vessels treated with PP1 could not maintain the constrictor response to NE over the

4 hr time period (Figure 6b; Table 1). At approximately 1 hr into the 4 hr time course

arterioles had returned to control diameters. The MEK inhibitor, PD 98059 (50 µM, n = 6)

similarly showed little effect on the spontaneous level of myogenic tone or the acute

contractile response to NE (Figure 6b; Table 1). However, in the presence of PD98059 the

contraction to NE waned over the 4 hr exposure period (Figure 6b; Table 1).

Time-dependent inhibition of the 4 hr NE constriction by the tyrosine phosphorylation

inhibitors prevented the possibility of determining the time to washout. Despite this

sufficient tone remained to assess adenosine (3 x 10-4 M) – induced dilation (Figure 7).

Control vessels exposed to NE for 4 hrs showed an impaired dilator response to adenosine

compared to the acute NE exposure while vessels treated with the inhibitors did not show an

attenuated adenosine response (Figure 7).


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Discussion:

The results of the present studies demonstrate that a 4 hr period of norepinephrine-

induced arteriolar constriction is sufficient to initiate phenomena that prolong the constrictor

response despite washout of the agonist. Such an effect was in contrast to that seen following

washout of an acute (5 min) NE -induced contraction. Further, the effect could not be

reproduced by a 4 hr exposure to KCl (75 mM) suggesting a possible requirement for

receptor binding and not contraction per se. Involvement of tyrosine phosphorylation is

suggested by inhibition of the potentiated adrenergic response by the broad spectrum tyrosine

kinase inhibitor, genistein, and the selective inhibitors PD98059 and PP1. As the impaired

relaxation response, following prolonged agonist exposure, was reversed by removal of

extracellular Ca2+ we have referred to the phenomenon as “functional remodeling”.

Control experiments were performed to demonstrate that changes in the ability to

relax following 4 hr NE treatment were not a function of either time or a pre-conditioning

phenomenon resulting from acute (5 min) exposure to the agonist. Thus, vessels showed

reproducible contractile responses to acute NE exposure applied at 4hr intervals and, further,

washout/relaxation was unaffected by this time period. A lack of a pre-conditioning effect

was shown by impaired relaxation to adenosine after 4hr continuous exposure to NE in a

group of vessels not subjected to any pre-exposure to the agonist. It was therefore concluded

that the impaired relaxation response was indeed a function of the prolonged agonist

exposure.

Remodeling has been used to describe, in particular, structural vascular adaptations

whereby alterations in the wall:lumen ratio are evident. Such remodeling can be hypotrophic,

eutrophic or hypertrophic depending on the growth response of the medial layer (27, 28, 29).

These classifications have resulted from the results of many earlier studies, especially those

related to changes in resistance vessel structure occurring in hypertension (1, 3, 14) and states


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of altered blood flow (6, 23). In the case of eutrophic remodeling it is believed that an initial

period of active vasoconstriction is followed by rearrangement of normal smooth muscle cells

to maintain a reduced diameter. Although not directly shown it is conceivable that the results

of the present study are consistent with an early stage in such a process. Interestingly, similar

signaling pathways (i.e. tyrosine phosphorylation events involving cSrc, and MAP) have been

implicated in both structural remodeling and the functional remodeling demonstrated in the

present studies (for example see references 20, 24, 25, 45). Similarly, support for early

agonist-induced remodeling events in tracheal smooth muscle involving tyrosine

phosphorylation has been provided by Gunst and colleagues (15, 42).

A possible explanation for the delayed relaxation response to the longer term

exposure to NE is that agonist binding initiates a pathway(s), in addition to Ca2+-calmodulin-

myosin light chain kinase mediated activation of myosin, that operate in a time-dependent

manner to influence the contraction/relaxation process. Such pathways may require either a

longer onset period, or time for washout, resulting in an altered time course in comparison to

those simply stimulated by a rise in Ca2+i (as might be expected for KCl-induced activation).

An obvious class of candidate mechanisms involves processes that alter the standard relation

between Ca2+ and contraction such as Ca2+ sensitization (40, 41). Ca2+ sensitization has been

demonstrated to occur in arteriolar smooth muscle and may involve tyrosine phosphorylation,

as tyrosine phosphatase inhibition, by pervanadate, causes constriction without a measurable

change in intracellular Ca2+ (31).

In addition to a Ca2+ sensitization process, a number of vasoactive agonists are known

to induce cytoskeletal rearrangement in various smooth muscle preparations (19, 20, 44, 45)

and modulate cell migration and attachment (20). Of possible relevance to our study, many

of these events have been shown to be dependent on tyrosine phosphorylation dependent

pathways. Of specific relevance to the present studies α1-adrenoceptor agonists, NE and


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phenylephrine, have been shown to activate tyrosine phosphorylation events including those

involving FAK, cSrc and p42 MAP kinase (2, 21, 47). Such studies have implicated these

signaling events in the adrenergic stimulation of non-selective cation currents, contraction

and mitogenic responses.

An interesting observation in the present study was that arteriolar constriction to KCl

(75 mM) was not maintained throughout the 4 hr observation period. Despite the continued

presence of KCl, arterioles had returned to control diameters by approximately 120 min. This

may indicate that maintained long-term constriction requires the involvement of signaling

mechanisms not activated by simple membrane depolarization. Similarly in the presence of

the non-selective tyrosine kinase inhibitor, genistein (10 and 30 µM), the NE constriction

tended to wane after approximately 60 - 90 minutes. This occurred despite genistein not

significantly affecting the acute contraction to NE (see Table 1). Thus it is tempting to

speculate that tyrosine phosphorylation – mediated events are required for maintenance of

prolonged arteriolar constriction. Further studies are required to determine whether these

events reflect the action of additional parallel contractile pathways (for example through thin

filament regulation, Ca2+ sensitization, cytoskeletal rearrangement etc) or form a support role

in maintaining the contractile pathways.

An obvious question relates to the physiological significance, or advantage, of an

apparent time-dependent switch in the mechanisms underlying a maintained arteriolar

contraction. It is conceivable that these additional pathways provide a mechanism where

there is maintained tension development at comparatively low energy cost. Such an

argument has previously been forwarded for the relevance of Ca2+ sensitization (40).

Regardless of the underlying mechanism this may confer an energetic advantage to smooth

muscles, such as are evident in the arteriolar wall, which exhibit a high level of maintained

tone.


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An additional consideration is that the functional and growth responses to

vasoconstrictors are often modulated by the generation of paracrine factors that exert either a

positive or negative effect on the vasomotor event. Of relevance to the results of the present

studies, DeFily et al. (11) have suggested that a component of the prolonged in vivo α1-

adrenergic-mediated constriction of canine coronary arterioles results from the release of

endothelin. Endothelin has been reported to modulate Ca2+ sensitivity via tyrosine

phosphorylation-dependent (35, 39) and independent (13) mechanisms in arterial smooth

muscle. Furthermore, prolonged in vivo administration of NE induces remodeling of small

arteries via a process dependent on endothelin (9). Consistent with these observations,

involvement of endothelin in the studies of DeFily was confirmed by experiments

demonstrating that either an ETA-receptor antagonist or an inhibitor of conversion of

prohormone to active endothelin prevented the sustained constriction to phenylephrine.

Interestingly, these investigators had previously shown that under in vitro conditions canine

coronary arterioles did not respond to α1-agonists suggesting that in vivo the source of

required endothelin was not restricted to the wall of the arteriole under study (22). It appears

likely that in the in vivo situation α1-agonist stimulation of cardiac myocyte endothelin

production contributed (42). With respect to the present data it was felt unlikely that in the

absence of significant parenchymal tissue, and in the presence of a superfusion system, that

sufficient quantities of a vasoconstrictor would accumulate to explain the prolonged

vasoconstrictor response.

In summary the results of the present study demonstrate that prolonged exposure of

arterioles to the contractile agonist NE leads to the stimulation of tyrosine phosphorylation-

dependent mechanisms which impair relaxation on washout of the agonist. Further, the

prolonged (over a 4 hr period) NE-induced contraction was totally prevented by inhibitors of

cSrc, MEK and general tyrosine phosphorylation suggesting that such events are critical to


19

force maintenance in arterioles. It is suggested that these observations are consistent with an

early phase of the remodeling process and, in particular, represent a functional adaptation

whereby force is maintained by mechanisms supporting, or acting in parallel to, Ca2+-

mediated contraction. Future experiments are required to determine whether these results are

applicable to prolonged myogenic constriction resulting from a sustained increase in

intraluminal pressure. In addition, to make these observations relevant to common clinical

situations, such as in hypertension, interactions that occur between mechanical and

neurohumoral stimuli should be considered.


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Acknowledgements:

Studies described in this manuscript were supported by grants from the National

Health and Medical Research Council of Australia, the National Heart Foundation the Sir

Edward Dunlop Foundation.


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American Journal of Physiology H00986-2002R2 Final Accepted Version

TABLE 1: Effect of Tyrosine Kinase Inhibitors and Demecolcine on Baseline Arteriolar

Diameter (% max) and Norepinephrine Responsiveness.

Baseline 5 µµµµM NE (5 min) 5 µµµµM NE (240 min)

49.1 + 0.4† 26.2 + 0.2Pre Genistein (n=4)Genistein (3 µM) 45.1 + 3.2 25.6 + 1.7 34.8 + 4.6 c

54.4 + 3.8 26.9 + 2.3Pre Genistein (n=4)Genistein (10 µM) 74.1 + 10.2 34.0 + 2.3 61.8 + 10.2 c

59.0 + 4.0 26.7 + 0.6Pre Genistein (n=4)Genistein (30 µM) 93.1 + 4.0 a 32.7 + 2.9 99.9 + 6.7 c

Baseline 5 µµµµM NE (5 min) 5 µµµµM NE (240 min)47.8 + 1.7 26.8 + 0.6 Pre PD98059 (n=6)

PD98059 (50 µM) 43.7 + 3.1 26.9 + 0.6 51.6 + 11.1c

Baseline 5 µµµµM NE (5 min) 5 µµµµM NE (240 min)Pre PP1 (n=4) 50.3 + 2.9 28.9 + 1.2PP1 (10 µM) 41.5 + 5.8 26.8 + 2.7 57.2 + 9.5 c

Baseline 5 µµµµM NE (5 min) 5 µµµµM NE (240 min)Pre Demecolcine (n=3) 54.5 + 5.8 24.9 + 0.5Demecolcine (10 µM) 39.6 + 2.8 a 11.9 + 1.7 b 17.8 + 2.6

† Mean ± SEM

a Significant (P<0.05) effect of drug treatment on baseline diameter (ANOVA).b Significant (P<0.05) effect of drug treatment on acute response to NE (ANOVA).c Significant (P<0.05) effect of drug treatment on prolonged response to NE (ANOVA).


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Figure Legends:

Figure 1: Example tracing illustrating the protocol used to contrast 5 min and 4 hr

exposures to contractile agents. The time scale has been omitted as a discontinuous scale has

been used in order to fit the entire protocol on one figure; 5 and 10 min time bars are shown

for the wash periods following acute and prolonged NE exposure, respectively.

Figure 2: The figure contrasts the 4hr constrictor responses to NE (5 µM, diamond

symbols, n = 10), KCl (75 mM, triangles, n=5) and control vessels exposed to buffer alone

(circles, n = 4). Results are presented as mean ± SEM; * P < 0.05 compared to baseline

(ANOVA).

Figure 3: Arteriolar smooth muscle Ca2+i changes during 4 hr exposure to NE (diamonds; n

= 7) as assessed using the Ca2+-sensitive dye fura 2 are shown in the lower panel. Time

controls (i.e. not exposed to NE) are shown by the circular symbols (n = 4). See Figure 2 for

diameter responses. Results are presented as mean ± SEM* P < 0.05 compared to baseline

(ANOVA).

Figure 4: Effect of NE (5 µM) exposure on subsequent dilator responses to topical

adenosine (3 x 10-4 M). Greater relaxation responses were seen after 5 min exposure to NE

(solid bars, n = 5) compared to that following 4 hr NE superfusion (open bar). This was not

a function of time as a repeat acute exposure to NE at 4 hr (n = 4) was not associated with an

impaired response to adenosine (hatched bar). Results are presented as mean ± SEM; * P <

0.05; n.s. p = 0.2344.

.


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Figure 5: Effect of the microtubule depolymerizing agent, demecolcine (10 µM), on 5

min and 4 hr exposure to NE (5 µM). Demecolcine caused vasoconstriction and a potentiated

constrictor response to NE without preventing the delayed relaxation response (to both

washout and adenosine) observed following 4 hr NE exposure. BL = baseline; Aden =

adenosine (30µM); DC = demicholcine (10µM); and 0 Ca = 0mM Ca2+/2mM EGTA buffer.

Wash refers to the diameter obtained after extensive washout of NE with fresh buffer as

described in Materials and Methods; 30 – 240 refers to selected timepoints (in minutes)

during the prolonged exposure to NE. A discontinuous scale time scale was used in order to

fit the entire protocol on one figure. Results are presented as mean ± SEM; n = 3. See Table

1 for statistical comparisons relating to the effect of demecolcine on diameter and NE

responsiveness.

Figure 6: Effect of tyrosine phosphorylation inhibitors on 5 minute and 4 hour exposure

to NE (5 µM). The upper panel shows the effect of increasing concentrations of genistein (n

= 4) on 5 minute and 4 hour exposures to NE while the lower panel contrasts the effects of

PP1 (n = 4), PD98059 (n = 6) and genistein (n = 4). Results are presented as mean ± SEM.

Figure 7: Effect of tyrosine phosphorylation inhibitors on the ability of adenosine (3 x

10-4 M) to elicit dilation following 4 hrs exposure to NE (5 µM) (open bars). Control

responses (i.e. in the absence of inhibitors) after 5 min NE treatment for each group are

shown in the solid bars. Results are presented as mean ± SEM. ** P < 0.01 compared to

relaxation following 5 min exposure to NE.


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