INTRODUCTION

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THE ABILITY OF THE ENDOTHELIUM to influence vascular diameter and vasoactive responses has been clearly demonstrated (10, 39). The specific mechanisms through which the endothelium exerts this influence may involve the release of nitric oxide (5, 14, 20, 31, 34, 35), vasodilatory prostanoids (12, 42), or hyperpolarizing factors, and is clearly dependent on the size of the vessel and vascular bed being studied. Therefore vascular patency may be understood as a delicate balance between constrictor and dilator mechanisms.

The question that we have chosen to address involves endothelial responses during vascular constriction. Multiple mechanisms may be involved in the release of endothelium-derived relaxing factors (EDRFs) during arterial constriction. Some studies have suggested the presence of endothelial ₁- or ₂-adrenergic receptors (3-5, 22). For example, Bockman and colleagues (3, 4) demonstrated significant [³H]rauwolscine (an ₂-adrenergic receptor antagonist) binding to pig aortic endothelial membranes and significant relaxation of rat superior mesenteric arteries to the ₂-adrenergic receptor agonist UK-14304 that was abolished after removal of the endothelium and inhibition of nitric oxide synthase (NOS). Boer and co-workers (5) demonstrated nitric oxide release by direct electrode measurement after constriction of isolated pulmonary arteries with the ₁-adrenergic receptor agonist phenylephrine; nitric oxide release was not observed with angiotensin II. This suggests the possibility of functional endothelial ₁-adrenergic receptors in pulmonary arteries. However, other mechanisms, not specifically linked to adrenergic receptor-mediated events, may also be involved. It was recently suggested (5, 14) that vascular smooth muscle (VSM) calcium can diffuse to the endothelium via myoendothelial cell junctions during arteriolar constriction, which leads to an increase in endothelial cell calcium and subsequent production of EDRFs.

Both endothelial -adrenergic receptor-dependent and -independent release of an EDRF during vascular constriction have been suggested from studies of arterioles in vivo (21, 22, 31, 33, 35). Our direct interest in this mechanism of vascular regulation was initiated by an observation while performing another study examining endothelial cell calcium (16). We recorded a rapid burst in endothelial cell calcium to an acute application of norepinephrine (NE, which is routinely used to verify vessel viability). Therefore the purposes of the present studies were to elucidate endothelial cell responses activated during adrenergic-mediated constriction of isolated arterioles from skeletal muscle, and furthermore to examine the potential intracellular mechanism we have measured and report endothelial cell calcium as a function of these adrenergic and adrenergic-independent constrictions.

	METHODS

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All animal-related techniques were approved by the University of Louisville Institutional Animal Care and Use Committee and conform to all federal, state, and local ethical standards.

Isolated vessel preparation. Experiments were conducted using arterioles isolated from the cremaster skeletal muscle of rats according to methods described previously (15-17). In brief, male Sprague-Dawley rats (250-300 g body wt) were anesthetized with a single concentration of veterinary thiopental sodium (100 mg/kg ip). The right cremaster muscle was excised and placed in a refrigerated (0-4°C) Lexan well containing MOPS in a physiological saline solution (MOPS-PSS; pH 7.4 ± 0.03) with 1% albumin (see Chemicals). A 2- to 3-mm segment of first-order (feed) arteriole devoid of branches was selected for microdissection. The arteriole segment was isolated, excised, and then cannulated with glass micropipettes. The preparation was then transferred to an inverted microscope and pressurized to its approximate in vivo pressure via a water manometer (90 cmH₂O) (28). Arterioles were initially passive (inner diameter 150-200 µm). During a subsequent 60-min equilibration period, the chamber that contained the arteriole was warmed to a temperature of 33.5 ± 0.5°C. Ordinarily at the end of this period the arteriole had gained some degree of spontaneous (intrinsic) tone (inner diameter 80-110 µm). Both the perfusion and the bath solutions consisted of MOPS-PSS without albumin. The criteria used for determining the viability of the isolated arteriolar preparation included development of spontaneous tone and reactivity to vasoactive substances such as NE (10⁷ M) and adenosine (Ade; 10⁴ M). Once these criteria were satisfied, the arteriole could remain viable for 4-6 h depending on the protocols used. The experiment was discontinued if any significant change in the ability of the arteriole to maintain spontaneous tone or react to the vasoactive substances was noted.

Video microscopy and digital image processing. A Nikon Diaphot 200 inverted microscope optimized for fluorescence was utilized for all studies. Observations were made with a Nikon fluorite ×20 (numerical aperture = 0.75) objective and/or a Nikon fluorite oil ×40 objective (numerical aperture = 1.30-0.80). A multi-image module (beam-splitting device) was connected to a Hamamatsu charge-coupled device (CCD) camera (red-field imaging) and a Hamamatsu intensified CCD camera system (fluorescence imaging). Arteriolar diameter was measured on-line with a video caliper device (Microcirculation Research Institute, Texas A&M University) and recorded with a MacLab chart recorder (ADInstruments).

Reactivity studies. Diameter was measured in separate randomized experimental runs to topically applied: the a nonspecific -adrenergic agonist NE (10⁹ to 10⁷ M), the endothelium-independent vasoconstrictor PGF₂ (10⁸ to 10⁶ M) (30), the endothelium-dependent vasodilator ACh (10⁷ to 10⁶ M), and the typically endothelium-independent vasodilator Ade (10⁴ M). After each agonist trial, the bathing solution was changed a minimum of three times and the arteriole was given 15 min to reequilibrate. In a separate series of experiments, arteriolar reactivity to luminal and abluminal application of NE and PGF₂ was examined.

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Calcium studies. Fluorescent images at excitation wavelengths of 340 and 380 nm were acquired every 5 s over a period of ~2 min. The first set of images within each trial represented control or baseline conditions. VSM cell calcium changes were evaluated in response to NE (10⁷ M), PGF₂ (10⁶ M), and ACh (10⁶ M). In similar, yet separate experiments, endothelial cell calcium was evaluated in response to NE (10⁷ M), PGF₂ (10⁶ M), ACh (10⁶ M), and Ade (10⁴ M). Finally, in yet another separate set of experiments, endothelial cell calcium was evaluated in response to the ₁-adrenergic receptor agonist phenylephrine (10⁷, 10⁶, and 10⁵ M) and the ₂-adrenergic receptor agonist UK-14304 (10⁷, 10⁶, and 10⁵ M). Separately, VSM calcium was also measured (16, 29) as a function of all of these agents for comparison.

Reagents and chemicals. The chemicals used were from Sigma unless noted otherwise. MOPS-PSS consisted of a solution made with (in mM) 145 NaCl, 5.0 KCl, 2.0 CaCl₂, 1.0 MgSO₄, 1.0 NaH₂PO₄, 5.0 dextrose (from Fisher), 2.0 pyruvate, 0.02 Na₂-EDTA, and 2.0 MOPS in PSS (pH 7.4 ± 0.03). One percent BSA (purity = 99.8%; from USB) was added to the MOPS-PSS to make the cold dissection solution. NE, ACh, Ade, TBA, and PGF₂ were brought into solution with MOPS-PSS. N^G-monomethyl-L-arginine (Calbiochem), N^G-monomethyl-D-arginine (D-NMMA, Calbiochem), N^G-nitro-L-arginine (L-NNA; Calbiochem), meclofenamate, and diclofenac were brought into solution with purified water, and then serial dilutions were made in MOPS-PSS. UK-14304 was brought into solution with DMSO and serial dilutions were made with MOPS-PSS. Arachidonic acid was brought into solution with 5% purified water-95% ethanol and was serial diluted in MOPS-PSS. MOPS-PSS with and without albumin was made before each experiment and stored either in the freezer or refrigerated until needed. All drug preparations were made fresh daily except for arachidonic acid, which was made into a 3 × 10² M stock solution and was stored under vacuum at 20°C until needed. All vehicles used to prepare the drug solutions were tested and did not alter vessel tone or intracellular calcium responses.

Statistics. Data are reported as means ± SE. Statistical analyses were carried out using the SuperAnova statistical analysis software (SAS Institute; Cary, NC). One-way and two-way ANOVAs were used to evaluate the diameter and calcium data where appropriate. Contrast-comparison tests were used to determine where the differences occurred. Statistical significance was assessed at P < 0.05 for all tests.

	RESULTS

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Removal of the endothelium. The mean baseline diameter of arterioles with an intact endothelium was 94 ± 1 µm, which was not significantly different from the baseline diameter of arterioles denuded of endothelium (94 ± 1 µm; Fig. 1). Confirmation of successful endothelium denudation was indicated by a reduced endothelial dilatory response to ACh (10⁶ M) without the loss of dilator capacity to Ade (10⁴ M) (see Fig. 1C). These findings are in agreement with our previous published data (15-17).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Maximal diameter responses of endothelium intact and denuded first-order isolated arterioles to norepinephrine (NE; A), PGF2 (B), and ACh and adenosine (Ade; C). Values are means ± SE (n = 6). §P < 0.05 vs. control; *P < 0.05 vs. endothelium intact condition.

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Inhibition of NOS. Baseline diameters were again not significantly different before and after incubation of the arterioles with the NOS inhibitor, L-NMMA (10⁴ M, 30 min; Fig. 2). Once again, the arteriolar constriction to NE after treatment with L-NMMA was significantly enhanced compared with the pretreated state (see Fig. 2A). L-NMMA treatment was without effect on PGF₂-induced constrictions (see Fig. 2B).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Maximal diameter responses of intact first-order isolated arterioles to NE (A), PGF2 (B), and ACh and Ade (C) in the absence and presence of NG-monomethyl-L-arginine (L-NMMA; 104 M, 30 min, n = 6), which inhibits nitric oxide synthase (NOS) production of nitric oxide (NO). Values are means ± SE; §P < 0.05 vs. control; *P < 0.05 vs. pretreatment.

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Cyclooxygenase inhibition. Once again, baseline arteriolar diameters before and after treatment with diclofenac (10⁶ M, 30 min), an inhibitor of cyclooxygenase, were not significantly different (see Fig. 3). Arteriolar reactivity to NE was not different between pre- and post-diclofenac-treatment conditions (see Fig. 3A). Similarly, no differences were observed in PGF₂-induced constrictions comparing before and after diclofenac treatments (see Fig. 3B).

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Maximal diameter responses of intact first-order isolated arterioles to NE (A) and PGF2 (B; n = 6) in the absence and presence of diclofenac (106 M, 30 min), an inhibitor of cyclooxygenase-mediated prostaglandin production. Maximal arteriolar diameter responses to ACh and arachidonic acid (AA) (C; n = 3) were also determined before and after treatment with diclofenac. Values are means ± SE; §P < 0.05 vs. control; *P < 0.05 vs. pretreatment.

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Inhibition of hyperpolarizing factor. Treatment of our isolated arteriolar preparation with TBA (5 × 10⁵ M, 30 min) had no effect on baseline control diameters (see Fig. 4). There was no significant difference in the arteriolar concentration-response reactivity to either NE, PGF₂, or Ade before or after incubation with TBA (see Fig. 4).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Maximal diameter responses of first-order isolated arterioles to NE (A), PGF2 (B), and ACh and Ade (C) in absence and presence of tetrabutylammonium (TBA; 5 × 105, 30 min, n = 5), a K+-channel blocker used routinely to ascertain the effects of hyperpolarizing factor(s). Values are means ± SE; §P < 0.05 vs. Control; *P < 0.05 vs. pretreatment.

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VSM calcium. VSM was successfully loaded with the calcium-sensitive fluorescent indicator fura 2 as previously described (29). Figure 5 illustrates the time course and peak VSM cell calcium changes to NE (10⁷ M) and PGF₂ (10⁶ M). All values after the addition of the pharmacological agent were significantly greater than control over the 100-s acquisition period (see Fig. 5A). Maximal VSM calcium in response to NE (10⁷ M) was 194 ± 14% of control with a concomitant arteriolar constriction of 63 ± 5% of control. Similarly, PGF₂ (10⁶ M) produced a maximal VSM calcium level of 144 ± 16% of control with a concomitant constriction of 67 ± 3% of control (see Fig. 5B).

View larger version (35K): [in this window] [in a new window]   Fig. 5.   A: vascular smooth muscle (VSM) cell calcium as a function of topical application of either NE or PGF2 versus time (n = 9). B: summated peak VSM calcium responses to the two constricting agents. Calcium data are represented as a percentage of control calculated from the F340/F380 ratio. Values are means ± SE; §P < 0.05 vs. control.

Endothelial cell calcium. Endothelial cells were successfully loaded with fura 2 as evidenced by a cellular axis that was longitudinally oriented along the arteriole in a spiraling arrangement as previously described (16, 17). NE (10⁷ M) significantly increased endothelial cell calcium, reaching 143 ± 10% of control (see Fig. 6A). Conversely, PGF₂ (10⁶ M) did not increase endothelial cell calcium above baseline levels. As expected, topical ACh (10⁶ M) produced a significant burst in endothelial cell calcium to 221 ± 26% of control (see Fig. 6B). Ade (10⁴ M) was without effect on endothelial cell calcium.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   A: arteriolar endothelial cell (EC) calcium responses (n = 6) to NE and PGF2. B: summated peak EC calcium responses to NE, PGF2, ACh, and Ade. Calcium data are represented as a percentage of control calculated from the F340/F380 ratio. Values are means ± SE. §P < 0.05 vs. control.

Adrenergic receptors. Topical application of the specific ₁-adrenergic receptor agonist phenylephrine yielded results similar to those already described for NE. Specifically, phenylephrine at concentrations of 10⁷ and 10⁶ M increased VSM calcium to 120 ± 14% and 212 ± 59% of control, respectively (see Fig. 7A) and produced significant arteriolar constrictions of 79 ± 3% and 53 ± 5% of control, respectively. Endothelial cell calcium did not rise above the control levels at the 10⁷ M phenylephrine concentration but did increase to 215 ± 26% of control with the 10⁶ M concentration (see Fig. 7B).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   A: maximal arteriolar VSM calcium responses (n = 4) to phenylephrine and UK-14304. B: maximal arteriolar endothelial cell calcium responses (n = 5) to phenylephrine (a specific 1-adrenergic agonist) and UK-14304 (a specific 2-adrenergic agonist). C: VSM cell calcium as a function of topical application of either phenylephrine or UK-14304 vs. time (n = 4). Calcium data are represented as the percentage of control calculated from the F340/F380 ratio. Values are means ± SE. §P < 0.05 vs. control.

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	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study has examined first-order arteriolar diameter, VSM calcium, and endothelial cell calcium as a function of either constrictor or dilator stimulation. The main findings from this study have clearly demonstrated that NE-induced arteriolar constriction is augmented after either the removal of the endothelium (see Fig. 1) or inhibition of NOS with L-NMMA (see Fig. 2). Similar results were not observed when arachidonic metabolism was inhibited via diclofenac (see Fig. 3) or when the actions of hyperpolarizing factor were inhibited with TBA (see Fig. 4). These results implicate the endothelium, and specifically nitric oxide, as a source of dilator capacity that acts to attenuate the constrictor effects of NE. These findings are in agreement with those of Sipkema and colleagues (5) who have published nitric oxide electrode measurements which suggest that during ₁-adrenergic receptor-mediated constriction of isolated pulmonary arteries, nitric oxide is released. In addition, endothelial cell calcium, which precedes the release of nitric oxide (8, 17, 20), increased in response to either NE (a nonspecific -adrenergic receptor agonist) or phenylephrine (a specific ₁-adrenergic agonist), but not to the -adrenergic-independent constrictor PGF₂ (30) (see Fig. 6) or the ₂-adrenergic agonist UK-14304 (see Fig. 7), which suggests that ₁-adrenergic receptors on these arterioles can function to increase endothelial cell calcium and the concomitant release of nitric oxide.

The elevation of endothelial cell calcium is known to increase the activity of the enzymes responsible for the production of vasoactive factors (9, 18), including dilators such as nitric oxide, prostacyclin, and hyperpolarizing factors (1, 8, 27). We report that endothelial cell calcium increased in response to NE, ACh (see Fig. 6), and phenylephrine (see Fig. 7). Therefore, any of these factors could have been involved in endothelial modulation of adrenergic tone. When we inhibited NOS with L-NMMA we found an increase in NE-induced constriction (see Fig. 2), which is consistent with results previously reported for isolated arterioles (2, 14). Because ACh-induced dilation of first-order cremaster arterioles involves increased endothelial cell calcium (16) via a nitric oxide-independent mechanism (2, 24), an additional EDRF might have been participating in the observed endothelial modulation of NE-induced constriction. To this end we examined the influence of cyclooxygenase products and hyperpolarization.

Cyclooxygenase inhibition has been reported to cause enhanced constriction in rabbit bronchial artery rings (42) and in the rat hindlimb vasculature (12). However, inhibition of cyclooxygenase was not reported to enhance sympathetic-mediated constriction of rat intestinal arterioles (32). Likewise we found no change in the NE-induced arteriolar constriction after cyclooxygenase inhibition with diclofenac (see Fig. 3). These results suggest that prostanoid involvement in adrenergically mediated contractile events may be species and/or vascular bed dependent.

The K⁺ channel blocker TBA was used to examine the possibility of a hyperpolarization influence during NE-induced arteriolar constriction. TBA has been reported to block calcium-activated K⁺ channels in rat muscle cells (40) and is also thought to inhibit ATP-sensitive K⁺ channels (36) as has been observed for other quaternary ammonium compounds (23). Although we did not know specifically where TBA was acting (endothelial and/or VSM cells), our primary goal was to inhibit the effects of hyperpolarization. Arterioles in the presence of TBA did not constrict differently in response to NE (see Fig. 4), which is similar to a previous finding in small skeletal muscle arteries (6). In addition, studies that have used techniques to block the release of cytochrome P-450 metabolites, which are known to cause smooth muscle hyperpolarization (19), also did not report any changes in NE-induced constriction (1, 7). These studies, taken together with our findings, suggest that an endothelial-derived hyperpolarizing factor is not involved in the reduction of -adrenergic-stimulated tone.

We have also reported that endothelial cell calcium increased in response to the ₁-adrenergic receptor agonist phenylephrine but not in response to the ₂-adrenergic receptor agonist UK-14304 (see Fig. 7). That UK-14304 did not induce a change in endothelial cell calcium within cremaster arterioles is consistent with findings in rat intestinal arterioles (33). Taken together, these results suggest that endothelial ₂-adrenergic receptors may not play a role in endothelial modulation of adrenergic constriction. However, endothelial ₂-adrenergic receptors are thought to induce nitric oxide formation in rat spinotrapezius arterioles and in larger arteries from other species (3, 39). Therefore, endothelial ₂-adrenergic receptor-dependent modulation of -adrenergic constriction is most likely dependent on the vascular bed studied.

Our results are based on the topical application of constrictor and dilator agents. It has been suggested that endothelial modulation of adrenergic constriction may in part be due to the metabolic breakdown of NE by endothelial cells (11, 38). In principle, the effective VSM concentration of luminally applied NE, compared with topical bath application, could potentially be reduced if the endothelium were acting as a significant metabolic barrier (11, 20). To address this possibility, we have made comparisons of arteriolar constriction to luminal or abluminal NE. We found no significant difference in steady state between the two application protocols in our isolated arteriolar preparation. It does appear that a rectifying barrier exists, because we are able to selectively load the endothelium with the calcium-sensitive fura 2 fluorescent dye from the lumen. This barrier is most likely selective based on size, charge, and lipophilicity. In our short-term experiments (usually <4 h in duration), we have not observed differences between luminal and abluminal application of the vasodilator and constrictor pharmacological agents.

We have also attempted to address the question regarding whether changes in the vascular dimensions commonly associated with vasoconstriction might contribute to the release of an EDRF. Recently, Kaley and colleagues (37) suggested that endothelial deformation may produce nitric oxide in arterioles from rat mesentery. We have explored a similar premise in these studies by examining endothelial cell calcium of arterioles isolated from rat skeletal muscle. We utilized PGF₂-induced constriction as a negative control for the vascular events. Our data demonstrate that the level of PGF₂-induced arteriolar constriction was not altered by any of the treatments to remove the effects of the various EDRFs. In addition, PGF₂ did not increase endothelial cell calcium (see Figs. 1-4 and 6). Similar confirmatory data are suggested by treatment with the ₂-adrenoceptor agonist UK-14304, which also produced an increase in VSM calcium and arteriolar constriction but did not alter endothelial cell calcium (see Fig. 7). Therefore, our data do not support the idea that constriction alone stimulates the release of vasodilators from the endothelium and that PGF₂ and UK-14304 were effective negative controls for the contractile event in these arterioles. Perhaps endothelial cell deformation is dependent on multiple facets of life such as vascular bed, arteriole size, or physiological factors such as flow, pulsatile motion, and/or shear stress (aspects not studied in our static flow conditions).

Gap-junctional proteins between the VSM and endothelium have been demonstrated in the hamster cheek pouch (25, 26). These published studies suggest that there may actually be exchange of ionic information via these junctional connections during constriction (14). During increases in VSM cell calcium, these connections might allow for diffusion of calcium from the muscle to the endothelium (14). Theoretically, any increase in smooth muscle cell calcium could lead to an increase endothelial cell calcium if myoendothelial cell junctions are present. Another more recent study from the same group demonstrates a phenylephrine-elicited vasoconstriction in hamster cheek-pouch arterioles that was preceded by an increase in VSM and endothelial cell calcium (41). We have attempted to explore this concept of a myoendothelial cell transfer event for calcium movement from VSM cell to endothelium by using agents that produce increases in VSM calcium yet act without direct effect on the endothelium. We utilized PGF₂ (30), which significantly increased VSM cell calcium (see Fig. 5) yet did not alter endothelial cell calcium (see Fig. 6). Moreover, increased VSM calcium and arteriolar constriction to the ₂-adrenergic agonist UK-14304 also did not produce any recordable increase in endothelial cell calcium (see Fig. 7). Previous reports have described another endothelium-independent constrictor mechanism, the myogenic response, which also increases VSM calcium (29) with no effect on endothelial calcium (15). However, our experiments must be interpreted with some degree of caution in that none of the endothelium-independent constrictors produced a transient burst in VSM calcium identical to ₁-adrenergic stimulation. Regardless of the differences in magnitude of the VSM calcium changes between ₁-adrenergic stimulation and those of PGF₂ and UK-14304, if myoendothelial cell junctions were freely flowing than any increase in VSM calcium should have produced some increase in endothelial cell calcium.

Alternatively, myoendothelial gap junctions could be regulated by some intracellular second messenger as has been suggested for cardiac gap junctions (13). Our current study as well as others (29, 41) have demonstrated a difference in the temporal pattern of VSM calcium with NE- or PGF₂-induced constriction. NE produced a large transient peak in VSM calcium, whereas PGF₂ produced a significant rise without a peak (see Fig. 5). This VSM transient is often referred to as the D-myo-inosital (1,4,5)-trisphosphate calcium peak and is thought to be mediated through the release of intracellular stores of calcium. Our data on endothelial cell calcium do not demonstrate an increase in calcium until after the NE-induced transient peak occurs in VSM calcium. Furthermore, in our studies, the sustained increase in endothelial cell calcium does not occur until after VSM levels begin to subside to steady-state levels not distinguishable in magnitude from the PGF₂ treatment (see Fig. 6A). These results are similar to those published by Yashiro and Duling (41), which demonstrate a transient peak in VSM calcium and a subsequent peak in endothelial cell calcium. Our data also demonstrate that UK-14304 produced a transient burst of VSM calcium (see Fig. 7C), albeit much less in magnitude that NE or phenylephrine, with the total absence of any observed increase in endothelial cell calcium (see Fig. 7). Nonetheless, second-messenger regulation may be a necessary component for myoendothelial communication resulting in the subsequent release of endothelium-derived relaxing factors.

Although our data do not refute the myoendothelial cell hypothesis, they do cast suspicion on another possible contributor to the endothelial dilator mechanisms during arteriolar constriction. Based on inference from our data presented here and data from the literature that suggest the presence of ₁- and ₂-receptors on endothelial cells (3-5, 22), one may speculate that ₁-adrenergic receptors on endothelial cells may function to increase cell calcium and result in promoting the action of NOS and the subsequent release of nitric oxide to produce an "escape" from adrenergic constriction. This "escape" phenomenon from adrenergic constriction may act to maintain at least some minimal level of blood flow to a given tissue providing at least a basic nutrient sustenance.