Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and alpha5 beta1 integrin ligands.

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.

RGDN peptide interaction with endothelial alpha5beta1 integrin causes sustained endothelin-dependent vasoconstriction of rat skeletal muscle arterioles.

The ability of an integrin-binding Arg-Gly-Asp-Asn (RGDN)- containing peptide to influence vascular tone by interacting with the alpha5beta1 integrin was studied using rat skeletal muscle arterioles. After blockade of beta3 integrin function, isolated arterioles with spontaneous tone showed concentration-dependent vasoconstrictions to topical application of GRGDNP, a peptide that shows a greater ability to interact with alpha5beta1 than with alphavbeta3. The constriction to GRGDNP (2.1 mM) was inhibited by blocking alpha5 integrin function, and was intensified by blocking beta3 integrin function. In contrast, GRGDSP, a peptide that interacts better with alphavbeta3, was unable to induce sustained constrictions. Removal of the endothelium abolished the vasoconstriction in response to GRGDNP, suggesting that the response was due to release of an endothelium-dependent factor. Indeed, blockade of ETA endothelin receptors with BQ-610 (1 microM), similar to removal of the endothelium and alpha5 integrin blockade, inhibited the vasoconstriction. These data indicate that interaction of RGD peptides, and in particular the RGDN sequence with endothelial cell alpha5beta1, causes endothelin-mediated arteriolar vasoconstriction. These results indicate that integrins are novel signaling receptors within the vascular wall that affect vasomotor tone, and may play an important role in vascular control.

Impaired arteriolar myogenic reactivity in early experimental diabetes.

Hyperperfusion and an increase in capillary pressure has been implicated in the pathogenesis of diabetic microangiopathy. The existence of such alterations suggests that the myogenic response to increased intravascular pressure may be altered in diabetes. To examine this, in vivo studies were performed on the rat cremaster muscle microcirculation of age-matched control and STZ-induced (65 mg/kg) diabetic rats (3-4 wk of diabetes). Anesthetized rats were enclosed in an airtight Plexiglas box with the cremaster muscle exteriorized into an organ bath containing Krebs' solution. To study myogenic responsiveness, box pressure was increased in steps of 10 mmHg from 0 to 30 mmHg for 2 min. Third-order arterioles of the control animals (lumen diameter 18 +/- 2 microns) responded to increased pressure with a rapid onset vasoconstriction. In contrast, the rate of development of the constriction was markedly attenuated in similar vessels (15 +/- 1 micron) of the diabetic animals, despite their ability to exhibit a similar maximal arteriolar constriction to that of the control animals. When 20 mmHg pressure steps were applied for only 10 s, arterioles of the diabetic animals constricted minimally, whereas those of the control animals constricted to 75% of the maximal response expected for that pressure increase (P < 0.01). Second-order arterioles of both groups of animals responded with a primarily passive distension to increased intravascular pressure suggesting that the impaired responsiveness of the third-order arterioles is not compensated for by an increase in the myogenic responsiveness of upstream vessels. Basal intravascular pressures, measured in first-, second-, and third-order arterioles, were similar in control and diabetic animals.(ABSTRACT TRUNCATED AT 250 WORDS)

A new paradigm for graduate research and training in the biomedical sciences and engineering.

98Emphasis on the individual investigator has fostered discovery for centuries, yet it is now recognized that the complexity of problems in the biomedical sciences and engineering requires collaborative efforts from individuals having diverse training and expertise. Various approaches can facilitate interdisciplinary interactions, but we submit that there is a critical need for a new educational paradigm for the way that we train biomedical engineers, life scientists, and mathematicians. We cannot continue to train graduate students in isolation within single disciplines, nor can we ask any one individual to learn all the essentials of biology, engineering, and mathematics. We must transform how students are trained and incorporate how real-world research and development are done-in diverse, interdisciplinary teams. Our fundamental vision is to create an innovative paradigm for graduate research and training that yields a new generation of biomedical engineers, life scientists, and mathematicians that is more diverse and that embraces and actively pursues a truly interdisciplinary, team-based approach to research based on a known benefit and mutual respect. In this paper, we describe our attempt to accomplish this via focused training in biomechanics, biomedical optics, mathematics, mechanobiology, and physiology. The overall approach is applicable, however, to most areas of biomedical research.

Transduction mechanisms involved in the regulation of myogenic activity.

Vascular smooth muscle has the ability to exist in a state of maintained partial constriction. This state of partial activation is initiated and/or maintained by the mechanical effects of distending pressure acting on the vascular wall. The intrinsic ability of vascular smooth muscle to respond to these mechanical forces is referred to as the myogenic mechanism. Within the past decade the signaling mechanisms responsible for mechanotransduction of myogenic phenomena have been the focus of extensive research. Two areas of active investigation include (1) the modulation of membrane ionic conductances by pressure/stretch and (2) the pressure/stretch-induced generation of second messengers known to be involved in vascular smooth muscle contraction. This review summarizes recent work aimed at understanding the mechanotransduction process in vascular smooth muscle.

Distributions of microvascular pressure in skeletal muscle of one-kidney, one clip, two-kidney, one clip, and deoxycorticosterone-salt hypertensive rats.

Studies were performed on the cremaster skeletal muscle in rats to investigate the microvascular changes that are associated with established one-kidney, one clip (1K1C) and two-kidney, one clip (2K1C) Goldblatt hypertension and with deoxycorticosterone (DOC)-salt hypertension. Rats were anesthetized with urethane and chloralose; and cremaster muscles with intact circulation and innervation were suspended in a controlled Krebs bath. Microvascular pressures and vessel diameters were measured at three consecutive arteriolar (A) and venular (V) branch levels. Arteriolar diameters (means +/- SEM) in normotensive (NT) rats were 119 +/- 7, 86 +/- 5, and 31 +/- 3 micron respectively for 1A, 2A, and 3A arterioles; and venule diameters were 218 +/- 12, 141 +/- 15, and 53 +/- 7 micron respectively for 1V, 2V, and 3V venules. As compared to NT rats, there was a selective decrease in lumen size (percent reduction from control) for 1A and 2A (23% to 38%) in 1K1C and 2K1C rats and for 1A, 2A, and 3A (42% to 44%) in DOC rats. Venule diameters were not significantly different between normotensive and hypertensive animals at any branch level. Femoral artery pressures were significantly elevated (greater than or equal to 43%) in all three forms of hypertension; however, this increase in pressure was not proportionally transmitted throughout the microcirculation. This was evidenced by normal pressure in 3A arterioles and in all venules for 1K1C and 2K1C rats and by normal pressures in 3V and larger venules for DOC rats. Our findings indicate that elevated arterial pressure in chronic renal hypertension is not transmitted uniformly across all microvascular segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoregulation and vasoconstriction in the intestine during acute renal hypertension.

The purpose of this study was to investigate whether local mechanisms of blood flow autoregulation mediate vasoconstriction during the early development of renal hypertension. Anesthetized rats were instrumented with Doppler flow probes on the celiac (CA), superior mesenteric (SMA), and renal arteries to measure flow velocity in these vessels. Acute two-kidney, one clip renal hypertension was produced by inflating a pneumatic occluder on the left renal artery to reduce flow velocity by 50%. Two hours after renal artery stenosis (RAS), femoral artery pressure (AP) was increased by 35%, CA resistance by 45%, and SMA resistance by 57%. No increases were observed in AP or in CA and SMA resistances for sham-operated, control rats. To determine if autoregulation contributed to the increase in SMA resistance, we protected the SMA vasculature from the increased arterial pressure by servocontrolled inflation of a pneumatic cuff implanted around the SMA. Although normalizing SMA pressure with the protective cuff significantly reduced (p less than 0.05) the increase in SMA resistance that occurred after RAS, SMA resistance remained elevated above control levels. These results suggest that (1) reduced intensity of SMA constriction produced by protection of the SMA is due to inhibition of a local autoregulatory mechanism that is contributing to the increase in SMA resistance during the acute development of renal hypertension, and (2) maintenance of elevated SMA resistance during protection from increased AP is the result of pressure-independent mechanisms that are activated subsequent to renal artery stenosis.

Hemodynamic characteristics of the intestinal microcirculation in renal hypertension.

This study investigated the microvascular changes that affect vascular resistance in the rat small intestine during two-kidney, one clip renal hypertension 4 weeks after renal artery stenosis. To study the intestinal microcirculation, a loop of the small intestine was exteriorized with intact circulation and innervation and a section of the bowel wall was prepared for observation with an intravital video microscopy system. Microvascular diameter, pressure, and flow velocity were measured for first, second, and third branch order arterioles and venules, using an image shearing monitor, servo-null micropipette system, and an optical Doppler velocimeter, respectively. The diameters of the first order arterioles and venules were significantly (p less than 0.05) reduced in hypertensive rats; however, diameters were unaltered in smaller second and third order arterioles and venules as compared with normotensive vessels. In hypertensive rats, mean arterial pressure was significantly (p less than 0.05) elevated (47%) and pressures also were elevated significantly (p less than 0.05) throughout the microcirculation, although by a proportionally smaller amount. Total network flow (i.e., first order arteriole flow) was significantly (p less than 0.05) reduced (40%) in hypertensive rats, but volume flows in individual second and third order arterioles were similar to flows measured in normotensive rats. Calculated total network resistance was increased (124%) in hypertensive rats. Thus, the intestinal microcirculation in rats with two-kidney, one clip renal hypertension is disturbed by elevated pressure and decreased total flow. The presence of normal flows in individual second and third order arterioles without any demonstrable difference in their diameters suggests that the predominant cause of elevated resistance across this segment of the intestinal microcirculation is a reduction in the number of perfused small arterioles.

Enhancement of histamine-induced vascular leakage by pertussis toxin in SJL/J mice but not BALB/c mice.

Pertussis toxin (PTX) from Bordetella pertussis is known to enhance inflammatory responses which involve histamine and serotonin, including cell-mediated delayed-type hypersensitivity reactions. In this study we examined the effects of PTX on histamine-modulated microvascular responses. The actions of histamine on arteriole diameter and post-capillary leaky site formation in the cremaster muscle were measured intra-vitally in two inbred strains of mice (viz. BALB/c and SLJ). In SJL mice the rate and extent of histamine-induced leaky site formation were greatly enhanced (from 8.3 to 21.0 leaky sites per 0.1 cm2) by pre-exposure to PTX. In sharp contrast, PTX did not alter histamine-induced leaky site formation in BALB/c mice. Histamine-mediated dilation in arterioles in both strains of mice were not enhanced by PTX. PTX may enhance the development of inflammatory responses by enhancing histamine-induced leaky site formation of the microvasculature.

Regional bioelectric properties of porcine airway epithelium.

Ion transport properties of pulmonary small airway epithelia are poorly understood. To characterize these properties, airways were excised from anesthetized pigs. Transepithelial potential difference (PD) and conductance were measured in five airway regions: trachea (T, 7.9 +/- 0.2 mm diam), mainstem bronchi (MB, 5.5 +/- 0.2 mm diam), large bronchi (LB, 1.69 +/- 0.12 mm diam), small bronchi (SB, 0.70 +/- 0.06 mm diam), and bronchioles (BR, 0.25 +/- 0.05 mm diam). T and MB were mounted in Ussing-type chambers, and LB, SB, and BR were cannulated with pipettes and perfused. PDs of control tissues were -9.7 +/- 0.8 mV (T), -4.0 +/- 0.5 mV (MB), -4.3 +/- 1.0 mV (LB), -4.5 +/- 0.4 mV (SB), and -1.5 +/- 0.4 mV (BR), lumen negative. Amiloride significantly (P < 0.05) inhibited PDs by 25-70% in all airway regions and decreased conductance 17-33% in all regions except LB where a 10% increase was observed. Bumetanide significantly reduced the amiloride-insensitive PD 54-62% in all regions except BR. Bumetanide had little effect on conductance in T, SB, and BR, but conductance was increased in MB and LB. All airways except the smallest BR significantly hyperpolarized when the solution that bathed the lumen was replaced with Cl(-)-free solution. In bronchioles, hyperpolarization by luminal Cl(-)-free solution was inversely related to fractional inhibition of PD with amiloride but directly related to lumen diameter. These results suggest that 1) porcine tracheas, bronchi, and bronchioles actively absorb Na+, and 2) secretion of Cl- may occur in all airway regions except small bronchioles.

Invited review: arteriolar smooth muscle mechanotransduction: Ca(2+) signaling pathways underlying myogenic reactivity.

The smooth muscle of arterioles responds to an increase in intraluminal pressure with vasoconstriction and with vasodilation when pressure is decreased. Such myogenic vasoconstriction provides a level of basal tone that enables arterioles to appropriately adjust diameter in response to neurohumoral stimuli. Key in this process of mechanotransduction is the role of changes in intracellular Ca(2+). However, it is becoming clear that considerable complexity exists in the spatiotemporal characteristics of the Ca(2+) signal and that changes in intracellular Ca(2+) may play roles other than direct effects on the contractile process via activation of myosin light-chain phosphorylation. The involvement of Ca(2+) may extend to modulation of ion channels and release of Ca(2+) from the sarcoplasmic reticulum, alterations in Ca(2+) sensitivity, and coupling between cells within the vessel wall. The purpose of this brief review is to summarize the current literature relating to Ca(2+) and the arteriolar myogenic response. Consideration is given to coupling of Ca(2+) changes to the mechanical stimuli, sources of Ca(2+), involvement of ion channels, and spatiotemporal aspects of intracellular Ca(2+) signaling.

On atomic force microscopy and the constitutive behavior of living cells.

Atomic force microscopy (AFM) is one of many new technologies available to study the mechanical properties and mechanobiological responses of living cells. Despite the widespread usage of this technology, there has been little attempt to develop new theoretical frameworks to interpret the associated data. Rather, most analyses rely on the classical Hertz solution for the indentation of an elastic half-space within the context of linearized elasticity. In contrast, we propose a fully nonlinear, constrained mixture model for adherent cells that allows one to account separately for the contributions of the three primary structural constituents of the cytoskeleton. Moreover, we extend a prior solution for a small indentation superimposed on a finite equibiaxial extension by incorporating in this mixture model for the special case of an initially random distribution of constituents (actin, intermediate filaments, and microtubules). We submit that this theoretical framework will allow an improved interpretation of indentation force-depth data from a sub-class of atomic force microscopy tests and will serve as an important analytical check for future finite element models. The latter will be necessary to exploit further the capabilities of both atomic force microscopy and nonlinear mixture theories for cell behavior.

In vitro hypoxia differentially affects constriction and relaxation responses of isolated pulmonary arteries from broiler and leghorn chickens.

Under normoxic conditions in vitro, isolated pulmonary arteries from broilers exhibit reduced endothelium-dependent relaxation responses when compared with Leghorns. In vivo, hypoxia increases the susceptibility of broiler chickens to pulmonary hypertension syndrome (PHS), whereas Leghorns are considered resistant to PHS. Because L-arginine supplementation decreases the incidence of PHS in vivo and improves the relaxation responses of broiler isolated pulmonary arteries in vitro, we hypothesized that in vitro hypoxia would further reduce the relaxation responses of broilers to endothelium-derived nitric oxide (EDNO)-dependent vasodilators and that L-arginine supplementation would alleviate this impairment. As a test of this hypothesis, pulmonary arteries from broiler and Leghorn chickens were isolated and exposed to normoxia or hypoxia in the presence or absence of L-arginine while their constriction and relaxation responses to vasoactive compounds were recorded. In broilers, hypoxia did not affect the constriction responses of isolated pulmonary arteries but decreased EDNO-dependent acetylcholine-induced relaxation responses. In contrast, in Leghorns hypoxia increased endothelin-1-induced vasoconstriction responses and reduced the EDNO-dependent relaxation responses only to the lowest concentration of acetylcholine used. L-Arginine supplementation augmented the relaxation responses to acetylcholine in broilers and Leghorns under normoxia but failed to augment them under hypoxia. Relaxation responses to the NO donor, sodium nitroprusside, were not affected by hypoxia in Leghorns but were increased by hypoxia in broilers. These results suggest that the increased incidence of PHS in broiler chickens reared under hypoxia may be associated with a hypoxia-induced reduction in the synthesis or activity of EDNO in the pulmonary circulation.

Time-dependent changes in smooth muscle cell stiffness and focal adhesion area in response to cyclic equibiaxial stretch.

Observations from diverse studies on cell biomechanics and mechanobiology reveal that altered mechanical stimuli can induce significant changes in cytoskeletal organization, focal adhesion complexes, and overall mechanical properties. To investigate effects of short-term equibiaxial stretching on the transverse stiffness of and remodeling of focal adhesions in vascular smooth muscle cells, we developed a cell-stretching device that can be combined with both atomic force and confocal microscopy. Results demonstrate that cyclic 10%, but not 5%, equibiaxial stretching at 0.25 Hz significantly and rapidly alters both cell stiffness and focal adhesion associated paxillin and vinculin. Moreover, measured changes in stiffness and focal adhesion area from baseline values tend to correlate well over the durations of stretching studied. It is suggested that remodeling of focal adhesions plays a critical role in regulating cell stiffness by recruiting and anchoring actin filaments, and that cells rapidly remodel in an attempt to maintain a homeostatic biomechanical state when perturbed above a threshold value.

A theoretical model for F-actin remodeling in vascular smooth muscle cells subjected to cyclic stretch.

A constrained mixture theory model was developed and used to estimate remodeling of F-actin in vascular smooth muscle cells that were subjected to 10% equibiaxial stretching for up to 30min. The model was based on a synthesis of data on time-dependent changes in atomic force microscopy measured cell stiffness and immunofluorescence measured focal adhesion associated vinculin as well as data on stress fiber stiffness and pre-stretch. Results suggest that an observed acute (after 2min of stretching) increase in cell stiffness is consistent with an increased stretch of the originally present F-actin plus an assembly of new F-actin having nearly homeostatic values of stretch. Moreover, the subsequent (after 30min of stretching) decrease in cell stiffness back towards the baseline value is consistent with a replacement of the overstretched original filaments with the new (reassembled), less stretched filaments. That is, overall cell response is consistent with a recently proposed concept of "tensional homeostasis" whereby cells seek to maintain constant certain mechanical factors via a remodeling of intracellular and transmembrane proteins. Although there is a need to refine the model based on more comprehensive data sets, using multiple experimental approaches, the present results suggest that a constrained mixture theory can capture salient features of the dynamics of F-actin remodeling and that it offers some advantages over many past methods of modeling, particularly those based on classical linearized viscoelasticity.

Novel genomic targets in oxidant-induced vascular injury.

To study the complex interaction between oxidative injury and the pathogenesis of vascular disease, vascular gene expression was examined in male Sprague-Dawley rats given 35 or 70 mg/kg allylamine, a synthetic amine converted to acrolein and hydrogen peroxide within the vascular wall. Vascular lesions and extensive vascular remodeling, coupled to increased production of 8-epi-PGF2alpha, nuclear localization of NFkappaB, and alterations in glutathione homeostasis, were observed in animals treated with allylamine for up to 20 days. Transcriptional profiling, immunohistochemistry, and in situ hybridization showed that genes involved in adhesion and extracellular matrix (ECM) (alpha(1) integrin, collagen), cytoskeletal rearrangements (alpha-smooth muscle actin, alpha-tropomyosin), and signal transduction (NFkappaB, osteopontin, and LINE) were altered by oxidant treatment. To evaluate mechanisms of gene dysregulation, cultured aortic smooth muscle cells were challenged with allylamine or its metabolites and processed for molecular analysis. These agents increased formation of reactive oxygen species and elicited changes in gene expression similar to those observed in vivo. Oxidative stress and changes in gene expression were inhibited by N-acetyl cysteine, a precursor of glutathione. These results indicate that genes along the ECM-integrin-cytoskeletal axis, in addition to LINE, are molecular targets in oxidant-induced vascular injury.

Responses of sequentially branching macro- and microvessels during reactive hyperemia in skeletal muscle.

Small artery and microvascular responses during reactive hyperemia were compared to determine which resistance-bearing vessels played a role in controlling blood flow and resistance for the cremaster skeletal muscle. Using an intravital video microscopy system, measurements of microvessel pressure, flow velocity, and diameter were obtained from cremaster muscles in anesthetized rats. These were compared with measurements of diameter that were obtained from the small arteries feeding the cremaster muscle. After a 60-sec occlusion of the sacral aorta, total cremaster blood flow increased approximately 28% and calculated microvascular resistance for the cremaster muscle fell 50%. During the period of occlusion, diameters of small arteries (159-292 micron) decreased despite the presence of smooth muscle tone. Likewise, the diameters of large arterioles (65-117 micron) decreased whereas small arterioles (16-30 micron) dilated. The decrease in diameter of the small arteries and large arterioles was accompanied by a significant fall in intravascular pressure, suggesting that the behavior of these vessels was largely passive. Immediately following the release of occlusion, small arteries and large arterioles returned to their control diameters while small arterioles remained in a dilated state for approximately 2 min. These results indicate that for the cremaster muscle, vascular responses vary along the length of the arterial tree during reactive hyperemia, small but not large arterioles are primarily responsible for the decrease in network resistance and subsequent hyperemia following occlusion, and the small feeder arteries did not dilate during reactive hyperemia but instead acted to set a limit on the decrease in network resistance and the increase in blood flow.

Vasoconstriction is amplified by autoregulation during vasoconstrictor-induced hypertension.

This study investigated the degree to which autoregulation of blood flow interacts with vasoconstrictors to determine vascular resistance. Anesthetized rats were instrumented with a Doppler flow probe on the superior mesenteric artery (SMA) to measure blood flow and for calculation of vascular resistance. An adjustable occluder was placed on the SMA to set local perfusion pressure at values independent of mean arterial pressure (MAP) even when MAP was increased by the vasoconstrictors. Infusion of angiotensin II (ANG II, 50-1,247 ng.kg-1.min-1) produced a dose-dependent rightward shift in the intestinal pressure-flow relationship and elevated MAP from 85 to 127 mmHg. Low doses of phenylephrine (PE, 2.5-12.4 micrograms.kg-1.min-1) failed to shift the pressure-flow curve but did increase arterial pressure from 83 to 102 mmHg. At higher doses (25-62 micrograms.kg-1.min-1), PE also shifted the pressure-flow curve to the right. Maintaining local perfusion pressure at different values during the infusion of ANG II or PE produced a family of dose-response curves, with each exhibiting a different maximum change in resistance. When local pressure was permitted to increase with MAP, the composite dose-response curve for resistance that was obtained reflected the influence of the rise in local pressure (i.e., auto-regulation) and vasoconstrictor dose. At low doses of PE the increase in vascular resistance was attributable solely to an autoregulatory response related to the rise in MAP and not due to the constrictor effects of PE. Thus these data indicate that the rise in MAP accompanying systemic infusion of a vasoconstrictor stimulates autoregulation to amplify the local increase in vascular resistance.

Cellular mechanisms involved in the vascular myogenic response.

By definition, the myogenic response is the contraction of a blood vessel that occurs when intravascular pressure is elevated and, conversely, the vasodilation that follows a reduction in pressure. Over the last several decades numerous investigators have demonstrated the importance of the myogenic response in the local regulations of blood flow, capillary pressure, and in the generation of basal vascular tone. Despite the considerable information obtained from these investigations, information about the cellular mechanisms that underlie this response has been slow to accumulate. Because of the physiological significance of the myogenic response, its mechanistic basis represents an important subject for research. Currently, there are several broad hypotheses concerning the sequence of events that couple changes in intravascular pressure or stretch with alterations in vascular smooth muscle activation. These hypotheses include 1) altered membrane properties leading to activation of ion channels; 2) modulation of biochemical cell-signaling pathways within vascular smooth muscle; 3) length-dependent changes in contractile protein function; and 4) endothelial-dependent modulation of vascular smooth muscle tone. This review summarizes current work relative to each of these hypotheses and describes a possible sequence of events to account for myogenic activation of vascular smooth muscle.