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The emerging role of Ca2+ sensitivity regulation in promoting myogenic vasoconstriction

Rudolf Schubert, Darcy Lidington, Steffen-Sebastian Bolz
DOI: http://dx.doi.org/10.1016/j.cardiores.2007.07.018 8-18 First published online: 3 January 2008


Growing evidence suggests that mechanisms which regulate the Ca2+ sensitivity of the contractile apparatus in vascular smooth muscle cells form the backbone of pressure-induced myogenic vasoconstriction. The modulation of Ca2+ sensitivity is suited to partially uncouple intracellular Ca2+ from constriction, thereby allowing the maintenance of tone with fully conserved function of other Ca2+-dependent processes. Following a brief review of ‘classical’ Ca2+-dependent signalling pathways involved in the myogenic response, the present review describes the emerging mechanisms that promote myogenic vasoconstriction via modulation of Ca2+ sensitivity. For the purpose of this review, Ca2+ sensitivity reflects the dynamic equilibrium between myosin light-chain kinase and myosin light-chain phosphatase activities in terms of its impact on vascular tone. Several signalling pathways (PKC, RhoA/Rho kinase, ROS) which have been identified as prominent regulators of Ca2+ sensitivity will be discussed. Although Ca2+ sensitivity modulation is clearly an important component of the myogenic response, attempts to integrate it into existing mechanistic models resulted in a two-phase model, with a predominant Ca2+-dependent ‘initiation/trigger’ phase followed by a Ca2+-independent ‘maintenance’ phase. We propose that the two-phase model is rather simplistic, because the literature reviewed here demonstrates that Ca2+-dependent and -independent mechanisms do not operate in isolation and are important at all stages of the response. The regulation of Ca2+ sensitivity, as an equal and complimentary partner of Ca2+-dependent processes, significantly enhances our understanding of the complex array of signalling pathways, which ultimately mediate the myogenic response.

  • Myogenic response
  • Small arteries
  • Calcium sensitivity

1. Introduction

More than a century ago, Sir William Bayliss described that ‘When [now] the pressure was raised inside the artery it was seen at first to swell, but immediately, and while the mercury was still kept at its height, a powerful contraction took place, in which the artery appeared to writhe like a worm. […] The reaction possessed by the arteries is of such a nature as to provide as far as possible for the maintenance of a constant flow of blood through the tissues supplied by them, whatever may be the height of the general blood pressure’.1 One hundred years and numerous investigations by cardiovascular physiologists later, we know about the widespread functional impact of this response on the cardiovascular system. Remarkably enough, despite all the collective efforts since the days of William Bayliss, our current understanding of the phenomenon remains rather incomplete. This review will briefly summarize the main aspects of what is presently known about the so-called ‘Bayliss effect’, with the main emphasis on recent additions that have considerably extended our knowledge regarding vascular smooth muscle contraction.

In his experiments, Bayliss had discovered an intrinsic property of smooth muscle cells, the myogenic response, which allows them to autonomously respond to changes in mechanical load. It should be noted that myogenic behaviour in small arteries includes two phenomena: myogenic tone (i.e., tone at a constant pressure level) and the myogenic response (i.e., the alteration of tone in response to a change in pressure).2 The myogenic response is, of course, capable of regulating blood flow within the microcirculation since (i) the vessel wall of flow-regulating arteries (resistance arteries or arterioles) is predominantly composed of smooth muscle cells and (ii) in the distal sections of the vascular tree, changes in mechanical load are a frequent phenomenon. Accordingly, myogenic responses have been described in several vascular beds (e.g. the mesenteric, skeletal muscle, cerebral, renal, and coronary circulation25). The description by Bayliss, which we have quoted to introduce this subject to the reader, focuses primarily on the local effect of the myogenic response in resistance arteries: the regulation of tissue perfusion. However, to lay the groundwork for a more complete understanding of this phenomenon, two other important functions of the myogenic response merit mention: (i) it contributes to the protection of adjacent capillary beds against fluctuations of systemic pressure and (ii) it feeds back to systemic blood pressure (which is a product of total peripheral resistance and cardiac output). The systemic effects of the myogenic response are prominent, and they significantly amplify (through a positive feedback mechanism) the immediate vasoconstrictor effects of pressor agents on systemic pressure.6

In summary, the ‘myogenic response’ mechanism is involved in virtually every aspect of resistance artery function. It contributes to the maintenance of arteriolar blood flow following an alteration of pressure load: an increase in transmural pressure will result in constriction of the artery; while a loss in pressure will be followed by dilation (Figure 1). Consequently, the relationship between arteriolar resistance and transmural pressure is proportional, a phenomenon that maintains capillary pressure within relatively constant limits.25 It is tempting to speculate that the serially arranged electrophysiological and biochemical signalling steps that comprise the ‘classical’ models of the myogenic response were originally inspired by the idea of a proportionally regulated system. However, there is growing evidence that the myogenic response is in fact the net sum of multiple parallel inputs (intra- and extracellular) and smooth muscle signalling processes.

Figure 1

An example of a typical myogenic response in a mouse resistance artery. The increase in transmural pressure in a side branch of the tibial artery from 40 mmHg to 80 mmHg led to an initial distension of the artery from 65 µm (left image; 40 mmHg) to 68 µm. During 5 minutes at 80 mmHg, the measured inner diameter decreased to 52 µm (right image; 5 min.; 80 mmHg). The two rectangles mark the regions of automated diameter measurement.

The aim of the present review is to describe the emerging mechanisms that promote myogenic vasoconstriction in response to acute changes in pressure (i.e., an ‘active’ myogenic response) through the regulation of Ca2+ sensitivity. To establish an effective framework for this discussion, the next section will briefly describe (i) current concepts with respect to how smooth muscle cells detect alterations in mechanical load; (ii) the primary intracellular signalling events that result; and (iii) the influences of other cells types (primarily endothelial and nerve cells) that can modulate the myogenic response. Confounding factors largely preclude the direct comparison of in vitro and in vivo models; in order to minimize ‘experimental’ contradictions, this review will primarily discuss in vitro evidence, with cautious comparison to in vivo results, where appropriate.

2. Conventional mechanisms

2.1 The stimulus

There is broad consensus that a change in arterial wall tension is the primary stimulus triggering the myogenic response.7 This conclusion is based on evidence that alterations in wall tension, but not vascular diameter (i.e., a change in cell length), correlate with the modulation of smooth muscle intracellular calcium levels and myosin light chain phosphorylation—two mandatory signalling requirements for smooth muscle contraction.8 According to this model, pressure-induced myogenic vasoconstriction abrogates its own stimulus (i.e., a change in vessel wall tension) through a negative-feedback mechanism that serves to limit the extent of the response.

2.2 The sensor

Many investigations have tried to identify a structure or mechanism to explain the translation of a physical stimulus into an electrical and biochemical cellular signalling event. Several sensors and mechanisms have been proposed, including stretch-activated cation channels,9 mechano-sensitive enzymes within the plasma membrane, and complex interactions between matrix metalloproteinases, the extracellular matrix, integrins and the cytoskeleton.1012 With regard to the cytoskeleton, there is substantial evidence for a regulatory role, but its exact nature is the subject of intense discussion.1317 At present, direct experimental evidence is lacking for many of the proposed sensor mechanisms (see discussion below and18). Rather than undertake the difficult task of assigning ‘primary’ and ‘secondary’ roles to these sensor mechanisms, we have chosen a more integrated approach that accepts that each mechanism shares in the regulation of the myogenic response, acting in either a sequential or parallel manner.

2.3 Intracellular signalling events; membrane depolarization

Although several hypotheses regarding the nature of pressure-induced depolarization have been proposed, they are largely based on rather indirect experimental evidence. The most popular hypothesis regarding the mechanism of depolarization is that smooth muscle cells possess stretch-activated cation channels, which carry an inward current that results in cell depolarization during the course of the myogenic response.19 Stretch-activated channels have been identified in freshly dispersed and cultured smooth muscle cells9,2023 and more recently, in isolated vessels.2427 However, the lack of specific blockers/inhibitors and the technical constraints of patch-clamp techniques have limited the direct experimental evidence for their physiological role in intact blood vessels. Recently, antisense oligonucleotide techniques have provided the first functional evidence that stretch-activated channels, specifically TRPC6, TRPM4 and possibly ENaC, are indeed involved in the myogenic response.24,25,28 It should be noted that the activities of voltage-dependent calcium channels29 and calcium-activated potassium channels30,31 may also be modulated by transmural pressure, and therefore these channels are additional candidates.

Calcium-activated potassium channels (K+Ca) carry a hyperpolarizing current proportional to the intracellular calcium concentration. An increase in K+Ca activity would result in inhibition of the myogenic response, a negative-feedback mechanism which may serve to limit the magnitude of the response. In accordance with this hypothesis, enhanced myogenic responses have been observed following K+Ca blockade, experimentally achieved with specific inhibitors of this channel.3234 K+Ca channels are known to be activated by ‘Ca2+ sparks’,35 whose frequency is regulated by transmural pressure.36 Another recently identified modulator of K+Ca channel activity is caveolin-1, a major structural protein stabilizing caveolae (a microenvironment critical for many signalling pathways). Genetic deletion of caveolin-1 increases K+Ca activity, thereby driving the membrane potential to more negative values.37 This results in the attenuation of myogenic responses.37 Whether caveolin-1 modulates K+Ca function through the stabilization of a microenvironment, or through a direct caveolin-1/K+Ca interaction, remains to be determined.

There are endogenous mediators, for example the cytochrome P-450 metabolite 20-HETE, that inhibit K+Ca38,39 and could, therefore, modulate the myogenic response.40 Indeed, the inhibition of cytochrome P-450 (and hence 20-HETE generation) has been shown to attenuate myogenic responses in several artery preparations.39,4144 It must be highlighted, however, that there is no direct evidence establishing linkages between the activities of (i) cytochrome P-450, (ii) K+Ca channels and (iii) the myogenic response. Further, there are no proposed mechanisms to explain pressure-dependent modulation of cytochrome P-450 activity.

For the sake of completeness, we must state that K+Ca do not appear to play a role in every vessel preparation;4547 and in at least one experimental setting, K+Ca blockade attenuated pressure-dependent depolarization and vasoconstriction.48 There is no clear explanation for the latter observation. These discrepancies need to be clarified in future investigations.

Since their activation increases an inward current, chloride channels may also participate in pressure-mediated smooth muscle cell depolarization.49 Unfortunately, chloride channel inhibitors are rather non-specific, and they also inhibit voltage-operated calcium channels50 and nonselective cation channels.19 Since these channels contribute to the myogenic response, the conclusions drawn from the use of chloride channel blockers have been limited. Recently, direct measurement of chloride fluxes has overcome these limitations, and alterations in transmural pressure indeed results in increased chloride fluxes,51 at least in some vessel preparations. However, the general inconsistency of the reported data makes it difficult to assign a specific role for these channels in the myogenic response.

Finally, it should be noted that voltage-gated potassium (Kv) channels have been investigated in the context of the myogenic response. These channels do not promote myogenic vasoconstriction per se, rather, they appear to provide a negative-feedback mechanism that limits depolarization in response to elevation of pressure and therefore limits myogenic vasoconstriction.5254

2.4 Intracellular signalling events: the transmembrane influx of Ca2+

Ca2+ influx via depolarization-dependent opening of voltage-operated calcium channels (VOCs), is widely considered to be the initial event of a signalling cascade that ultimately increases myosin (MLC20) phosphorylation and hence, promotes vasoconstriction. VOCs are logically assumed to respond to alterations in membrane potential, although there is evidence that these channels can also directly respond to stretch.29 Most investigations, however, clearly indicate that VOC channels are responding to pressure-induced changes in membrane potential.25 From a teleological standpoint, VOC-mediated Ca2+ influx, which can be rapidly and proportionally adapted to alterations in pressure via the membrane potential, is an ideal mechanism to explain why the myogenic response occurs in both directions. While there is little doubt that the myogenic response depends heavily on VOCs, in particular, Cav1.2,55,56 residual responses have occasionally been detected following VOC inhibition, indicating that VOCs may not be the only channel mediating pressure-induced Ca2+ entry.57 Although the influx of Ca2+ through potential-independent calcium channels must be considered an alternative route for Ca2+ entry, e.g. via capacitive calcium entry routes,47,58,59 there is minimal data available to advance this hypothesis within current models of the myogenic response.

Questions regarding the temporal and causal relationships between membrane depolarization and Ca2+ influx have been addressed by several studies. Blockade of transmembrane Ca2+ influx (via inhibition of VOCs by pharmacological means) abolishes pressure-induced vasoconstriction with minimal impact on membrane depolarization.60,61 Complementing these observations are experiments where pressure-induced depolarization was inhibited (via clamping of the membrane potential with high extracellular potassium): despite the loss of pressure-induced membrane depolarization, myogenic responses persisted.29 The potential dissociation between these two cellular events would have several implications: (i) Ca2+ ions appear to play a rather limited role in pressure-mediated depolarization; (ii) membrane depolarization is not a mandatory signal for pressure-induced vasoconstriction; (iii) signalling pathways acting in parallel to membrane depolarization must exist; (iv) mechano-transduction mechanisms independent of stretch-activated channels likely exist; and (v) second messenger-like processes may play an important role in the initiation of the myogenic response. The generation of pro-constrictive metabolites in response to pressure could also provide an element of inertia, which could help to explain why the myogenic response persists despite the abrogation of its own primary stimulus (wall tension).

As previously illustrated, an increase in intracellular Ca2+ (Figure 2) is a mandatory prerequisite for vasoconstriction in response to elevation in transmural pressure. The experimental basis for this conclusion is very broad, and the results from different vascular beds in different species are quite coherent. There are two primary mechanisms that elevate intracellular Ca2+: (i) transmembrane influx and (ii) release from intracellular stores. The most common experimental strategy to study the relevance of intracellular Ca2+ stores is to deplete them (via ryanodine receptor activation) prior to increasing pressure. In several vascular preparations, intracellular Ca2+ store depletion does not impact the myogenic response,29,62,63 making internal Ca2+ release and sequestration appear to be relatively minor contributors to the myogenic response. In contrast, there is solid experimental evidence for a significant role of transmembrane influx of Ca2+ following elevation of transmural pressure. Removal of extracellular Ca2+ prevents the pressure-induced increase in intracellular Ca2+,8,60 the increase in MLC20 phosphorylation,8,64 and results in complete inhibition of the myogenic response. Because of its importance for the myogenic response, the mechanisms that trigger and maintain Ca2+ influx from the extracellular space have been intensively studied.

Figure 2

Pressure-induced alterations in smooth muscle Ca2+ and vascular diameter in hamster resistance artery. In arteries isolated from the hamster gracilis muscle (4th generation side branch of the femoral artery, 180 µm resting diameter), a stepwise change in transmural pressure from 45 mmHg to 110 mmHg (upper panel) induces a rapid increase in smooth muscle intracellular Ca2+ (middle panel), determined using the Ca2+-sensitive dye Fura 2 (fluorescence ratio F340 nm/F380 nm). The lower panel shows the associated change in diameter, which displays the typical biphasic response. The passive distension of the vessel that immediately follows the pressure step is reversed by a continuous, active vasoconstriction. Of note, the myogenic response seen in this artery is weaker than the one in Figure 1, possibly reflecting differences in artery size, vascular bed and species that all contribute to the nature of the myogenic response.

Although pressure may stimulate a prominent elevation of intracellular Ca2+, vasoconstriction does not necessarily follow (e.g., as shown in rat mesenteric arteries65). We noted this phenomenon in hamster skeletal muscle resistance arteries, and were able to unmask pressure-stimulated vasoconstriction by inhibiting nitric oxide (NO) synthase(s) with L-NA. To complement this observation, we have also demonstrated that the NO donor sodium nitroprusside abolishes myogenic vasoconstriction without effect on pressure-induced Ca2+ entry.66 Obviously, the (pressure-induced) increases in smooth muscle cell Ca2+ and resultant contractile responses do not necessarily possess a linear relationship. As a consequence, descriptions of the myogenic response will remain incomplete without an understanding of the mechanisms that could serve to explain this non-linear behavior.

The preceding example also highlights the fact that diffusible factors (e.g., NO) have the potential to modulate the myogenic response. Therefore, our overview of what is currently known about the myogenic response would not be complete without a short description of possible extrinsic influences. In this regard, the vascular wall provides an environment where smooth muscle cells closely interact with two other cells types: endothelial cells and nerves/nerve endings. The close proximity could allow these cells to modulate the myogenic response through paracrine actions on smooth muscle cells. In general, endothelial and neuronal cells minimally influence the myogenic response.67,68 Yet, exceptions where the myogenic response is enhanced following endothelial denudation clearly exist.6971 It is unclear, however, whether the endothelial cells in these cases are constitutively active or subject to pressure-mediated regulation.

To conclude, despite a rather large investigative effort, our understanding of the complex underlying mechanisms that regulate the myogenic response remains incomplete. Although Ca2+ clearly plays a crucial role in mediating the myogenic response, recent evidence clearly supports the existence of an additional level of regulation, one that determines the magnitude of smooth muscle cell contraction for a given alteration in intracellular Ca2+. These mechanisms have been intensively evaluated in recent years. As a result of advances in this field of study, the novel concept of Ca2+ sensitivity regulation, which complements Ca2+-dependent mechanisms, has been integrated into current models of myogenic vasoconstriction. The second section of this review is dedicated to this topic.

3. Novel mechanisms

A common denominator of the mechanisms presented above is their association with intracellular Ca2+ alterations. However, as already mentioned, alterations in intracellular Ca2+ can not fully explain the myogenic response. A closer look at the simultaneous diameter and Ca2+i measurements in Figure 2 reveals that myogenic vasoconstriction gains strength despite declining levels of Ca2+i. In other words, this phase of the response is characterized by increasing vasoconstriction at a constant level of Ca2+i. We believe that the delay between the immediate increase in calcium and full vasoconstriction reflects a continuous increase in calcium sensitivity. The modulation of Ca2+ sensitivity is an emerging concept, which has only recently been extensively reviewed by the Somlyos.72 Their definition of the phenomenon states that ‘Ca2+ sensitivity of smooth muscle and nonmuscle myosin II reflects the ratio of activities of myosin light-chain kinase (MLCK) to myosin light-chain phosphatase (MLCP) and is a major, regulated determinant of numerous cellular processes’. In this regard, the ‘lag phase’ observed in Figure 2 may result from the ongoing process of establishing a new equilibrium between the activities of MLCK and MLCP.

3.1 Regulation of actin/myosin interaction in smooth muscle cells of resistance arteries

As we know today, the arterial constriction that Bayliss observed in response to elevated pressure results from an increased actin–myosin interaction in smooth muscle cells. This interaction provides the force necessary for the relative movement of both filaments that macroscopically appears as a shortening of smooth muscle cell length. Because of their circumferential orientation within the arterial wall, the shortening of multiple smooth muscle cells translates into vessel constriction.

To precisely regulate vessel diameter, the interaction between actin and myosin must be tightly controlled. The decisive molecular switch, MLC20 (the 20 kDa myosin light chain), is located on the myosin head, which is the protein domain that actually interacts with actin. Phosphorylation of this switch favors actin–myosin interaction, whereas interaction is prevented in its non-phosphorylated state. The dynamic equilibrium between phosphorylated and non-phosphorylated MLC20 is governed by two enzymes, myosin light chain kinase (MLCK) and myosin light-chain phosphatase (MLCP). MLCK is primarily regulated in a Ca2+-dependent manner, i.e., all effectors that impact smooth muscle intracellular Ca2+ indirectly effect MLCK activity. Indeed, the majority of studies investigating the myogenic response have focused on mechanisms that involve the regulation of intracellular Ca2+ and hence, MLCK activity.25 Although elevation of intracellular Ca2+ is mandatory for the myogenic response (i.e., pharmacological blockade of Ca2+ influx abolishes myogenic responsiveness), there is growing evidence that signalling mechanisms independent of Ca2+ are integral components of this complex response. These more recently described mechanisms act primarily through regulation of MLCP, allowing for changes in MLC20 phosphorylation at a constant level of MLCK activity. Such alterations in Ca2+ sensitivity provide, for the first time, a mechanistic solution to the enigmatic observation that vasoconstriction continues despite unchanged calcium levels in the post-initiation phase of the myogenic response (Figure 2).

Relying on the definition provided by Somlyo and Somlyo,72 the appropriate measure of Ca2+ sensitivity would be the degree of MLC20 phosphorylation at a constant level of intracellular Ca2+ (and in principle, MLCK activity), through an alteration in MLCP activity. Unfortunately, the routine measurement of MLCP activity and/or MLC20 phosphorylation was not feasible in the small artery investigations reviewed here. In these studies, changes in the relationship between intracellular Ca2+ and vessel diameter (or wall tension) served as a surrogate marker for alterations in Ca2+ sensitivity.

Of note, other potentially relevant mechanisms have been labelled ‘Ca2+-independent’, including alterations in the relationship in myosin phosphorylation and vessel diameter (i.e., thin filament regulation73,74) or long-term structural adaptations of the vascular wall. These mechanisms will not be addressed in the present review, since to our best knowledge, they have not been investigated in the context of the myogenic response or are beyond the scope of the present review. Of course, this does not preclude that they contribute to myogenic vasoconstriction.

3.2 Evidence for the involvement of Ca2+-independent mechanisms in the myogenic response

Before delving into molecular signalling cascades, we feel it is important to briefly review the key observations that indicate the existence of Ca2+-independent components within the myogenic response. The relatively recent development of Ca2+ sensitive fluorescent dyes (e.g., Fura-2) combined with their integration into isolated artery models, allowed for the first measurements that dynamically related changes in intracellular smooth muscle Ca2+ and vessel diameter. The advent of this technique lead to the ground-breaking observation that hamster cheek pouch arterioles display augmented myogenic responses to increasing pressure-step amplitudes (e.g., comparing 40–60, 40–100 and 40–140 cm H2O step changes), but possess similar pressure-induced Ca2+ elevations.75

Ratiometric Ca2+ dyes (e.g., Fura 2) are typically distributed homogenously throughout the cytosol and thus conventional imaging techniques cannot determine local or compartmentalized alterations in Ca2+. Although confocal microscopy could provide the necessary spatial resolution for localized Ca2+ measurement, it lacks the necessary ultraviolet excitation wavelengths and the rapid switching between them for true ratiometric measurement. The limitation sparked debate as to whether subcellular fluctuations in Ca2+ exist (i.e., near the actin-myosin interaction points) that are not detectable with global Ca2+ measurements. More modern techniques now possess the spatial resolution necessary to measure Ca2+ concentrations at highly localized subcellular domains, and have therefore allowed the investigation of this hypothesis. To date, the only subcellular fluctuations in Ca2+ detected in myogenically active vessels are ‘Ca2+ sparks’ that are localized at the plasma membrane.35 These sparks, however, do not appear to promote myogenic vasoconstriction. Therefore, there is currently no evidence for changes in Ca2+i (neither local nor global) that would explain the observations of D'Angelo et al.75

In addition, by exposing isolated vessels to high extracellular K+, Lagaud and co-workers effectively overcame the issue of fluctuations in subcellular Ca2+.76 Their treatment depolarized the smooth muscle cells, opened L-type Ca2+ channels, and allowed for free transmembrane flux of Ca2+. Under these conditions, Ca2+i can be adjusted by Ca2+ex, i.e., Ca2+i can be externally clamped. Lagaud and coworkers observed that application of 60 mmol/L KCl effectively clamped Ca2+i in isolated arteries to a level above that observed in response to pressure under non-depolarized conditions. Despite this ‘clamp’ of Ca2+i, elevation of transmural pressure in the same arteries continued to evoke myogenic vasoconstriction.76 A caveat must be applied to this conclusion, since high extracellular KCl not only increased Ca2+i, but also vascular tone. How alterations in these parameters prior to the pressure-step per se modified the characteristics of the myogenic response has not been addressed. Indeed, Ratz and coworkers77 have recently documented that high extracellular KCl markedly augments the Ca2+ sensitivity of the smooth muscle cell contractile apparatus. Therefore, the applied conditions could have promoted Ca2+-independent mechanisms that are not normally so prominent in response to elevated pressure. Nevertheless, these observations are consistent with the idea that myogenic responses possess a Ca2+-independent component.

VanBavel et al. noted that in rat mesenteric small arteries elevation of pressure induces a passive distention of the vessel in addition to myogenic vasoconstriction. They proposed that these two components, passive distension and active constriction, individually impact the Ca2+i-diameter relationship when they are integrated.78 As a consequence, they defined vascular tone in terms of wall tension, rather than vessel diameter. Specifically, the actual wall tension was normalized to the maximal active tension that the vessel could develop at the same diameter.

Applying their definition of tone, the Ca2+ sensitivity (i.e., the Ca2+-tone relationship) following elevation of pressure was quantified relative to KCl-induced alterations in tone, and found to be higher.7880 Their approach, however, hinges on the assumption that KCl-mediated changes in tone are entirely Ca2+-dependent, with constant Ca2+ sensitivity. As we have previously noted, this assumption may not be correct,77 and thus the authors have potentially measured the differential potency of two Ca2+-sensitizing agents. Nevertheless, these observations contribute to our understanding of Ca2+ sensitization during the myogenic response.

The measurement of the Ca2+i-tone relationship at different transmural pressures offers yet another means to assess Ca2+ sensitivity. In a recent study employing rat tail small arteries, the Ca2+i-tone relationship was determined at 10 mmHg and 80 mmHg, and found to be significantly higher at the latter.81 This approach, which is well suited to isolating the effect of pressure under otherwise constant conditions, also strongly supports a role for Ca2+ sensitization in the myogenic response.

It should be mentioned that permeabilization of smooth muscle cells by the insertion of membrane pores (e.g., β-escin or α-toxin) is widely employed to assess Ca2+ sensitivity. The advantage of this approach is that it eliminates ion fluxes across the plasma membrane, provides buffering against localized Ca2+i changes and effectively clamps Ca2+i. However, permeabilization dramatically alters the plasma membrane's biophysical properties and its linkage to intracellular signalling cascades. It is not surprising therefore, that permeabilization of isolated vessels disrupts the myogenic response.29

Based on the solid evidence, it is now accepted that changes in Ca2+ sensitivity contribute to the myogenic response. Consequently, the primary focus of the field has now turned to elucidating the cellular signalling pathways that mediate pressure-induced Ca2+ sensitization.

3.3 Indications for the involvement of PKC

Since PKC is a recognized upstream modulator of processes that regulate Ca2+ sensitivity,72 it has been targeted by several studies in order to ascertain its role in the myogenic response. Inhibition of PKC ablates myogenic vasoconstriction in arteries derived from several vascular beds, without impact on pressure-induced Ca2+ elevation.65,76,79 However, research efforts related to the involvement of PKC have been limited by several factors, including (i) the existence of several functionally distinct PKC isoforms; (ii) poor specificity of chemical inhibitors, and (iii) the non-uniform expression and distribution of PKC throughout the vascular tree. Further, assuming a prominent role for PKC in maintaining Ca2+ sensitivity under resting conditions, its inhibition could potentially render the vessel unresponsive to pro-constrictive stimuli. As a consequence, PKC inhibition could abolish myogenic vasoconstriction without direct involvement in pressure-induced signalling.79

To avoid the technical challenges associated with the impact of basal PKC activity on Ca2+i-diameter relationships, current investigations have focused on more direct indicators of PKC activation in response to pressure. To this end, increased generation of diacylglycerol (an endogenous activator of PKC)82 and the translocation of PKC-α to the plasma membrane (a marker of PKC activation)83 have been demonstrated in response to elevated transmural pressure. With respect to the observed translocation of PKC-α,83 the temporal kinetics of the translocation following elevation of pressure were consistent with its involvement in the myogenic response. Monitoring of temporal kinetics is highly enlightening, especially when one considers that pharmacological inhibitors (e.g. the MAP-kinase inhibitor PD98059) often possess limited specificity.84 Monitoring of temporal kinetics has excluded the participation of several pressure-sensitive signalling pathways, specifically tyrosine kinases85 and p42/44 MAP kinase,86 because they do not possess the correct temporal kinetics to mediate myogenic vasoconstriction. The pressure-dependent activation of these signalling pathways may be, at least in the particular vessels studied, involved in cellular responses unrelated to myogenic tone (e.g., growth or remodelling).

PKC inhibition has been shown to promote increases in Ca2+i in a pressure-dependent manner through interactions with voltage-dependent Ca2+ channels87 and other cation channels.26 However, in some vessels, PKC inhibition does not affect the myogenic response.81,88 This highlights the variability in individual components targeted by PKC in response to pressure across species and vascular beds. Therefore, it is imperative to confine conclusions regarding the modulation of myogenic responses by PKC to the actual vascular bed and species studied. Nevertheless, the body of evidence reviewed here suggests a role of PKC as a Ca2+-sensitizing element within the regulation of the myogenic response.

3.4 Indications for the involvement of RhoA/Rho kinase

Although RhoA has been shown to modulate Ca2+ sensitivity in permeabilized smooth muscle cells,72,73 it was not until the recent emergence of specific inhibitors (e.g., Y27632) against its main downstream target, Rho kinase, that its contribution to the myogenic response could be investigated in intact arteries. Since this time, the use of Y27632 has confirmed an important regulatory role of Rho kinase in myogenic vasoconstriction, although the results have been quantitatively diverse. For example, in rat mesenteric80 and hamster gracilis arteries,89 Rho kinase inhibition abolishes myogenic vasoconstriction without impact on pressure-induced elevation in Ca2+, while in rat tail81 and cerebral small arteries,90 it attenuates myogenic vasoconstriction with concurrent augmentation of pressure-induced Ca2+ elevation. However, a direct assessment of Ca2+ sensitivity, via measurement of the Ca2+i-tone relationship, has consistently yielded lower sensitivity during Rho kinase inhibition.80,81

While Rho kinase activation is often tightly associated with activation of RhoA,72 the conclusions drawn from the use of Y27632 must be strictly confined to Rho kinase, as the extrapolation upstream to RhoA is highly indirect and speculative. Only recently, genetic targeting of the RhoA signalling, through transfection of activity-modulating mutants of RhoA and Rho kinase, has confirmed a prominent role for both of these proteins within the context of the myogenic response.89 Appropriately, the inhibition profile for both the RhoA- and Rho kinase-inhibiting genetic constructs matched that of Y27632, in that they abolished myogenic vasoconstriction without effecting pressure-induced Ca2+ elevation.89

As with PKC, the issue of relative contributions of constitutive versus pressure-induced Rho kinase activity is a challenge to overcome. To address this problem, the Y27632-sensitive (i.e., Rho-kinase-dependent) fraction of the MR was compared at low and high transmural pressure.81 Indeed, Y27632 had a more pronounced effect at 80 mmHg than at 10 mmHg, providing indirect evidence for a pressure-dependent activation of Rho kinase in small arteries. Translocation of RhoA to the plasma membrane, a marker of its activation, in response to mechanical stimuli has been shown in both an isobaric91 or an isometric vessel preparation.92 Caveolae have been identified as a possible target microdomain of RhoA translocation,91 suggesting that it clusters with other signalling components in a highly regulated manner.

Because RhoA/Rho kinase signalling is a prominent regulator of MLCP activity (and hence Ca2+ sensitivity) in vascular smooth muscle cells, its involvement in regulating the myogenic response is not surprising. Obviously, this conclusion is primarily based on the use of inhibitory strategies; direct confirmation in small resistance arteries has proven to be methodologically challenging.

3.5 Indications for the involvement of reactive oxygen species

The involvement of reactive oxygen species (ROS) in the modulation of Ca2+ sensitivity and the myogenic response is a relatively new revelation. The initial indication that O2 modulates the myogenic response was that arteries from transgenic animals lacking NADPH oxidase function (knockout of p47phox and transgenic for dominant-negative N17Rac) display no myogenic activity.93 This investigation was later complemented by observations that mice deficient in superoxide dismutase (SOD; an endogenous antioxidant enzyme that metabolizes superoxide to H2O2) possessed enhanced myogenic reactivity.94 The use of chemical NAPDH oxidase inhibitors and antioxidants appeared to clinch a mandatory role of O2 generation for myogenic reactivity.

In addressing the mechanistic basis for these observations, Keller et al. found that specific inhibition of NADPH oxidase with an inhibitory peptide reduced the myogenic response but did not affect pressure-induced increases in Ca2+.95 They, therefore, concluded that ROS played a modulatory, rather than an obligatory role in the regulation of Ca2+ sensitivity during myogenic vasoconstriction. Keller et al. suggested that the discrepancy between their investigation and that of Nowicki et al. lies in the profiles of the different inhibitors employed (chemical, peptide and genetic in nature). The majority of the protocols apparently fail to specifically inhibit NADPH oxidase, and possess wide-ranging non-specific effects (e.g., a profound reduction of the pressure-induced increase in Ca2+) that affect the experimental outcome.95 This is a particularly illustrative example that, as already noted for PKC and RhoA, non-specific effects of ‘putatively-specific’ inhibition strategies can seriously compromise the conclusions drawn.

3.6 Integration of Ca2+ sensitivity into current models of the myogenic response

The original modeling of the myogenic response occurred before the concept of Ca2+ sensitivity modulation emerged. As such, these models were primarily based on an assumption that the relationship between Ca2+i and constriction remains static. The myogenic response, therefore, was proposed to result solely from pressure-induced Ca2+ influx, which subsequently activated the downstream targets calmodulin and MLCK to enhance MLC20 phosphorylation and ultimately increase constriction.

However, there is no doubt today that modulation of Ca2+ sensitivity is an important component of the myogenic response and its integration into a new mechanistic model has profoundly changed the way we view this complex response (for a simplified overview see Figure 3). Initially, Ca2+-sensitizing mechanisms were simply added to the previous Ca2+-dependent model. This resulted in a two-phase model, with a predominant Ca2+-dependent ‘initiation/trigger’ phase followed by a Ca2+-independent ‘maintenance’ phase. The integration of this ‘maintenance’ phase into the model provided an explanation for the ongoing constriction despite decreasing Ca2+i. While providing a basic mechanistic framework, this two-phase model remains rather simplistic because it implies that Ca2+-dependent and -independent mechanisms work in virtual isolation, with relative importance at different time points during the response.

Figure 3

Signalling pathways in vascular smooth muscle reported to contribute to the myogenic response. Schematic representation depicting the primary signalling pathways that are currently believed to initiate and maintain the myogenic response. Of significance, the represented pathways (e.g., ‘Ca2+-dependent’ (left) and ‘Ca2+-independent’ (right)) are not mutually exclusive. In fact, there is growing evidence that the myogenic response possesses a high degree of redundancy at the mechanosensor and intracellular signalling pathway levels. For details, please, refer to the main text. Abbreviations used in the scheme: TRP – transient receptor protein cation channel, ENaC – degenerin/epithelial sodium cation channel; Cav1.2 – voltage‐operated Ca2+ channel; CCE – capacitative calcium entry; (Ca2+)4CAM – Ca2+-calmodulin complex; MLCK – myosin light‐chain kinase; MLC20 – myosin light-chain regulatory domain (20 kDa); Sk1 – sphingosine kinase 1; PLC – phospholipase C; GPCR – G-protein-coupled receptor; IP3 – inositol triphosphate; DAG – diacylglycerol; PKC – protein kinase C; IP3R – IP3 receptor; RyR – ryanodine receptor; sER – smooth endoplasmic reticulum; CYP4A – cytochrome P450 4A; MLCP – myosin light-chain phosphatase.

Based on the data reviewed here, this current model does not reflect the true nature of the myogenic response. Clearly, Ca2+-dependent and -independent mechanisms are both absolutely critical to the myogenic response, since inhibition of either component has the potential to abolish it. The two-phase model was partially based on the hypothesis that the Ca2+ elevation in response to elevated pressure was a mechanical response and therefore a much faster signalling event than the second messenger cascades that ultimately terminate in modulation of Ca2+ sensitivity. However, recent evidence involving the role of sphingosine kinase 1 (Sk1) in the modulation of the myogenic response suggests otherwise. In isolated hamster resistance arteries, Sk1 signalling is known to enhance Ca2+ sensitivity by a RhoA/Rho kinase-dependent mechanism.89 However, Sk1 signalling also possesses clear immediate effects on pressure-induced Ca2+ elevation.89 Thus, it was concluded that Sk1 signalling (presumably via its metabolite sphingosine-1-phosphate) orchestrates both components of the myogenic response as an upstream modulator.

An inherent logic of this conclusion is that both components are acting simultaneously. In favour of this conclusion, Lagaud et al.76 observed immediate myogenic vasoconstriction under conditions of clamped Ca2+i. Since Ca2+i could not change under these conditions, the entire response must have been mediated through an immediate enhancement of Ca2+ sensitivity. Of note, this does not contradict the conclusion that Ca2+ elevation is mandatory, since in these experiments, the Ca2+i required for sufficient MLCK activity was provided by the KCl-dependent depolarization employed to clamp Ca2+i at an elevated level. The observations of Lagaud et al.76 also suggest that a rapid Ca2+ elevation is not likely to act as a ‘trigger’ for Ca2+ sensitizing mechanisms, as one might predict in the context of a two-phase response.

4. Conclusions

Because of the pleiotropic and ubiquitous effects of Ca2+, its intracellular concentration is tightly controlled and preferentially kept at a low level. Therefore, from a teleological standpoint, mechanisms that regulate Ca2+ sensitivity, rather than Ca2+i are advantageous to the cell. The interplay between these pathways may have important functional consequences: considering that small arteries and arterioles are permanently exposed to transmural pressure, these microvessels are in a perpetual state of myogenic activation. In the long-term, shifting the control of vasoconstriction from Ca2+-dependent to Ca2+-independent mechanisms would allow these vessels to maintain the appropriate tone to withstand transmural pressure, and also keep intracellular Ca2+ low to avoid interference with other Ca2+ dependent processes.

Indeed, several signalling pathways (PKC, RhoA/Rho kinase, ROS) serve the purpose of regulating Ca2+ sensitivity. Although identified to participate in the regulation of the myogenic response, the complex issue of cross-talk between these signalling pathways merits further investigation. Superimposed on the issue of cross-talk, differences between species and vascular beds must also be taken into account as the fundamental models are devised. It is clear that certain criteria must be fulfilled in order to confidently include the involvement of a particular signalling molecule/pathway in the regulation of Ca2+ sensitivity during the myogenic response: (i) there must be direct measurement of pressure-induced activation of the molecule/pathway; (ii) its activation must possess an appropriate temporal parallel to the contractile response; (iii) its activation must either (a) not affect the pressure-induced elevation in Ca2+i or (b) be unaffected by the clamping of Ca2+i; and (iv) be inhibited by specific inhibitors or by genetic inhibition with dominant negative mutants or knockout models. Although the individual studies discussed in this review did not simultaneously test for all of these elements, taken together, they present evidence comprising all of the listed compulsory requirements.

Conflict of interest: none declared.


This work was supported by Deutsche Forschungsgemeinschaft grant SCHU 805/7-1 (RS) Deutsche Forschungsgemeinschaft collaboration grant 444 KAN 113/1/06 (RS + StSB); start-up funding from the Department of Physiology and the Heart and Stroke/Richard Lewar Centre of Excellence at the University of Toronto (StSB); a research grant from the Heart and Stroke Foundation of Ontario (NA6198; StSB); and an International Opportunities Program Seed Grant from the Canadian Institutes of Health Research (OPD-83184; StSB + RS).


  • This article was published online by Elsevier on 3 August, 2007.


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