Pressure-induced myogenic tone and role of 20-HETE in mediating autoregulation of cerebral blood flow

Abstract

While myogenic force in response to a changing arterial pressure has been described early in the 20th century, it was not until 1984 that the effect of a sequential increase in intraluminal pressure on cannulated cerebral arterial preparations was found to result in pressure-dependent membrane depolarization associated with spike generation and reduction in lumen diameter. Despite a great deal of effort by different laboratories and investigators, the identification of the existence of a mediator of the pressure-induced myogenic constriction in arterial muscle remained a challenge. It was the original finding by our laboratory that demonstrated the capacity of cerebral arterial muscle cells to express the cytochrome P-450 4A enzyme that catalyzes the formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid, the production of which in cerebral arterial muscle cells increases with the elevation in intravascular pressure. 20-HETE activates protein kinase C and causes the inhibition of Ca²⁺-activated K⁺ channels, depolarizes arterial muscle cell membrane, and activates L-type Ca²⁺ channel to increase intracellular Ca²⁺ levels and evoke vasoconstriction. The inhibition of 20-HETE formation attenuates pressure-induced arterial myogenic constriction in vitro and blunts the autoregulation of cerebral blood flow in vivo. We suggest that the formation and action of cytochrome P-450-derived 20-HETE in cerebral arterial muscle could play a critically important role in the control of cerebral arterial tone and the autoregulation of cerebral blood flow under physiological conditions.

stepwise increase of transmural pressure in isolated and cannulated cerebral arteries gives rise to a series of signaling events that culminate in stepwise constriction of the arterial segments. These arterial muscle signaling events include membrane depolarization, reduction in outward K⁺ current, increase in inward Ca²⁺ current (via L-type Ca²⁺ channels), translocation of PKC and phosphorylation, and elevation of inositol 1,4,5-trisphosphate and diacylglycerol, all of which are related to the activation of arterial muscle and allow arterial diameter to track changes in blood pressure, thereby maintaining blood flow relatively constant (3, 5, 19, 25, 27–31, 44). The signaling pathway mediating these events involves the production of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) (15, 21, 23, 26, 34). These series of signaling reactions maintain blood flow constant despite wide fluctuations in arterial pressure. The reasons for such maintenance of blood flow can be explained both physiologically as well as teleologically. The skull does not allow for changes in vascular volume or flow because of the physics of incompressible fluids in a confined space. The space available for the distribution of volume is necessary for changes in the vascular space and protection of the microcirculation from changes in blood pressure (2, 18). Similarly, changes in intravascular shear forces act to modify vascular diameter, thereby altering myogenic capacity and total intravascular volume (45, 46). The action of shear is considered limited to actions emanating from endothelial factors, whereas the action of pressure acts on both endothelial and arterial muscle cells, exerting influences that act to both constrict and dilate (19, 22, 44, 46). While myogenic forces in response to a changing arterial pressure have been described early in the 20th century (3), it was not until 1984 (19) that we observed that increasing intravascular pressure depolarized the vascular wall, altering sensitivity to vasoactive agents via evoking changes in ionic conductance systems compared with the then standard practice of mounting ring segments on wires connected to tension recording myographs (6). Depicted in Fig. 1 is the effect of a sequential increase in intraluminal pressure on cannulated cerebral arterial preparations that resulted in pressure-dependent membrane depolarization and reduction in lumen diameter.

Fig. 1.

I: series of photomicrographs of cat cerebral arterial segments taken at different transmural pressures. The horizontal dashed lines in each photo are placed at the internal borders of the vessel wall. The numbers in the left-hand corner of each photo give the membrane potential (E_m) recorded in the artery at the time the micrograph was taken. II: actual chart records of intracellular E_m recorded from muscle cells of isolated cerebral arteries at 4 different transmural pressures [10 mmHg (A), 100 mmHg (B), 140 mmHg (C), and 170 mmHg (D)]. Sequentially, increase in transmural pressures caused a marked pressure-dependent decrease in internal diameter and membrane depolarization associated with spike generation (19).

Release of Arachidonic Acid from Membrane-Bound Phospholipid Pools and Generation of Arachidonic Acid-Derived Intermediates: Control of Cerebral Arteriole Tone by Acting on K⁺ and Ca²⁺ Channels

The stimulation of different types of lipases can catalyze hydrolytic cleavage and release arachidonic acid (AA) from the sin-2 position of membrane phospholipids (23, 44, 52, 53). This enzymatically released AA can be metabolized to prostaglandins, leukotrienes, and, as mainly discussed in this review, to the cytochrome P-450 (CYP) (40, 41, 50)-catalyzed metabolic products (7, 16, 23, 42, 47). Numerous communications and many awards have been given for the discovery of the vascular CYP monooxygenases (CYP-ω-hydroxylase and CYP-epoxygenase), which metabolize AA to 20-HETE and epoxyeicosatrienoic acids (EETs), respectively, and the soluble epoxide hydrolases that inactivate the EETs (9, 12, 14–16, 21, 47). The major CYP 4A-ω-hydroxylase metabolite of AA 20-HETE was first identified and detected in intact cerebral arteries in 1994 by the laboratories of Harder et al. (Fig. 2B) (21), and its specific production was latter confirmed in isolated cerebral arterial muscle cells (16) (Fig. 2A). 20-HETE was found to be one of the most potent vasoconstrictors yet identified. The reported physiological actions of the CYP metabolites of AA including 20-HETE are via modification of ion channel functions (9, 14, 16, 17, 21). 20-HETE was found to inhibit the activity of the large-conductance Ca²⁺-activated K⁺ channel (K_Ca) and to increase the influx of Ca²⁺ through the activation of cerebral arterial L-type Ca²⁺ channels (16, 21, 31). In the late 1980s and early 1990s, we found that the production of 20-HETE mediates the myogenic response, representing a major discovery at this time (21, 26, 34). It was found that 20-HETE had a profound action to depolarize and raise intracellular Ca²⁺ in the vascular wall through its action on arterial K_Ca and L-type Ca²⁺ channels (Figs. 3 and 4II) (16, 21, 31).

Fig. 2.

Analysis of 20-hydroxyeicosatrienoic acid (20-HETE) formation in cerebral arterial muscle. I: reverse-phase HPLC chromatograph illustrating CYP 4A ω-hydroxylase metabolism of [¹⁴C]arachidonic acid ([¹⁴C]AA) to 20-HETE in isolated cerebral arterial muscle cells [control (A), standard (B), and 17-octadecynoic acid (17-ODYA; C)]. CPM, counts per minute; EETs, epoxyeicosatrienoic acids. II: identification of 20-HETE by gas chromatography-mass spectroscopy. Refer to details in Refs. 16 and 21.

Fig. 3.

Single-channel large-conductance Ca²⁺-activated K⁺ (K_Ca) currents (I_KCa) recorded at patch potential 0 mV from cerebral arterial muscle cells using 4.7 mM K⁺ in the bathing solution and 145 mM K⁺ in the recording pipette solution. A: when compared with control, treatment with the cytochrome P-450 4A (CYP 4A) ω-hydroxylase inhibitor 17-ODYA (20 μM) induced an increase in single-channel activity (middle) that was inhibited by subsequent addition of 1 nM 20-HETE (bottom). C, closed; O, open. B: changes in the number of events and channel open state probability (NP_o) following treatment with 17-ODYA or 20-HETE, which induced activation and inhibition, respectively, of a 217 pS K_Ca single-channel current. *P < 0.05; n = 5 to 6 cells (21).

Fig. 4.

I: effects of 1 μM 20-HETE on intracellular Ca²⁺ level in arterial smooth muscle cells isolated from renal arteries. Bar graph depicts mean response of isolated arterial muscle cells to 20-HETE (1 μM) in 8 separate experiments. *P < 0.05 compared with control values (34). I, inset: increase in intracellular Ca²⁺ level following stimulation of the arterial muscle cells with 20-HETE. II: activation of macroscopic L-type Ca²⁺ channel current recorded from cat cerebral arterial muscle cells by exogenously applied 20-HETE (100 nM) (A). 20-HETE significantly increased the magnitude of peak inward L-type Ca²⁺ current (B) recorded during 10-mV step depolarization between −50 to +50 mV (16).

Role of PKC and the Mechanism of Action of 20-HETE in Cerebral Vascular Control

At this point in time we could only speculate that an increase in arterial pressure activated phospholipases via a stretch by the stimulation of molecules in the vessel wall (11, 23, 39, 44). Even though we do not know the precise mechanism through which pressure induces the production of 20-HETE to initiate the myogenic response, we do know that PKC and perhaps inositol 1,4,5-trisphosphate are involved (11, 25, 29, 30, 39). The vasoconstriction action of pressure can be linked to depolarization (19, 22) subsequent to the release of 20-HETE and the activation and translocation of PKC (15, 21, 26, 31, 34). Stimulus-induced activation of phospholipase C, including via the action of pressure, is linked to the subsequent formation of diacylglycerol, AA, and its metabolites, which activate PKC (23, 39), inhibit K_Ca activity, and depolarize the plasma membrane, leading to the elevation of intracellular Ca²⁺ concentration and activation of cerebral arterial muscle (16, 21, 31, 34). Indeed, the dominant action of 20-HETE appears to be through the activation of PKC (31). Major supporting evidence for this hypothesis is the finding that the inhibition of endogenous PKC using selective N-myristoylated PKC pseudosubstrate inhibitor peptide [MyrΨPKC-I_(19–27)] blocks 20-HETE-induced reduction of K_Ca current recorded in cerebral vascular smooth muscle cells (Fig. 5) without imposing an independent action (31). Similarly, PMA, a prototype activator of PKC, induced the inhibition of K_Ca current in the same cerebral arterial muscle cells that was attenuated by the actions of the above-mentioned PKC pseudosubstrate inhibitor peptide, which is mimicked by 20-HETE (31). The fact that MyrΨPKC-I_(19–27) does not change whole cell K_Ca current by itself compared with control indicates the involvement of PKC-mediated pathways in the 20-HETE-induced inhibition of K_Ca current. Furthermore, biochemical data demonstrate that 20-HETE increases the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS), a sensitive and selective endogenous substrate and indicator of the level of PKC activity (Fig. 6), providing supportive evidence for the role of PKC as one of the molecular targets for the action of 20-HETE.

Fig. 5.

Inhibition of PKC attenuates the inhibition of whole cell K⁺ currents by 20-HETE. A: application of the N-myristoylated PKC pseudosubstrate inhibitor peptide [MyrΨPKC-I_(19–27); 100 nm] did not alter whole cell K⁺ current (middle), and 20-HETE (300 nm) failed to reduce whole cell K⁺ current in the presence of this PKC inhibitor (bottom). B: averaged peak current-voltage relation before (○), after addition of MyrΨPKC-I_(19–27) (100 nm; ●), and after addition of 300 nm 20-HETE in the continued presence of 100 nm MyrΨPKC-I_(19–27) (▼). 20-HETE did not reduce whole cell K⁺ current amplitude following PKC inhibition (n = 5 for each experiment). Vertical lines represent SE (31).

Fig. 6.

Concentration-dependent inhibition by MyrΨPKC-I_(19–27) of 20-HETE (1 μM)-induced phosphorylation of 87 kDa myristoylated alanine-rich C kinase substrate (MARCKs) protein in cat cerebral arterial muscle cells. A: representative autoradiogram depicting the concentration-related inhibitor effects of MyrΨPKC-I_(19–27) on 20-HETE-induced phosphorylation of MARCKs. B: line graph illustrating the summary of data from n = 6 such experiments run in parallel. Mean data were fitted with a single exponential function (31).

Important Role of 20-HETE in Pressure-Induced Myogenic Autoregulation

Many actions have been ascribed to effects of 20-HETE (15, 16, 31, 47). One of the most important of these is the activation of PKC-induced phosphorylation (31). This action of 20-HETE results in the inhibition of K_Ca channel activity and membrane depolarization. Such membrane depolarization is responsible for a variety of signaling events, including the activation of L-type Ca²⁺ channels and an increase in inward Ca²⁺ current. We believe the membrane depolarization in response to the inhibition of K_Ca channel activity creates a positive driving force for Ca²⁺ influx and a very potent signal for cell activation. These findings have been duplicated in numerous publications (15, 16, 31, 47) and is a signature event in pressure-induced activation of cerebral arterial muscle as transmural pressure increases (16). We also feel that this could also be one of the initiating mechanisms resulting in pressure-induced activation of cerebral arterioles. The pivotal role of the activation of PKC in the 20-HETE-induced K_Ca channel inhibition and membrane depolarization was convincingly confirmed by the ability of 20-HETE to increase the phosphorylation of the MARCKS, a specific and sensitive endogenous target for phosphorylation induced by PKC activation, and by the similarity of the 20-HETE-induced inhibitory action on the K_Ca channel to that induced by the selective PKC activator PMA (31; and references therein). It is presumed that such activation of PKC-associated MARCKS phosphorylation signaling pathway underlying the vasoconstrictor actions of 20-HETE could be a link for the interaction with other endogenous signaling molecules or genes containing a MARCKS domain and has the potential to influence membrane ionic events regulating myogenic responses in the cerebral circulation or in other vascular beds. Thus the 20-HETE PKC and MARCKS signaling pathway could be considered as a crucial signaling cascade for the assessment of brain function in health and/or in cerebrovascular disorders under different disease conditions.

Opposing Actions of CYP-Derived EETS on Pressure-Mediated Activation of 20-HETE: Ying-Yang Actions of Astrocytes and Possible Role in Functional Hyperemia

The expression of CYP 2C11 epoxygenase and the formation of EETs in primary cultures of astrocytes derived from cortical and hippocampal regions of the rat brain were first demonstrated in our laboratory by molecular and biochemical methods (1). Nearly a decade later, a second CYP epoxygenase isoform named CYP 4X1 was identified by Bylund et al. (7) in our laboratory in the rat, mouse, and human brain by reverse transcription polymerase chain reaction. CYP 4X1 was shown to be expressed in neurons throughout the brain, brain stem, hippocampus, cortex, and cerebellum, as well as vascular endothelial cells (7). Unlike 20-HETE, EETs increase the activity of K_Ca channel currents and hyperpolarize cerebral and other arterial beds, leading to vasodilation (9, 14, 17, 20). In the last 15 years since the findings of CYP 2C11 epoxygenase in astrocytes, a tremendous amount of work form numerous laboratories has led to the identification of many biological functions associated with EETs. Thus EETs have been shown to play a role in vascular cell proliferation, angiogenesis, anti-inflammation, antinociception, neuroprotection, and most importantly in vascular reactivity where they function to mediate functional hyperemia (9, 10, 14, 17, 43, 47, 49, 54, 57–59). Pressure-induced activation of cerebral arteries sets the stage for hyperemic increases in blood flow as EETs potently dilate the cerebral microcirculation supplying blood and oxygen to metabolically needy neurons. This scenario is depicted in Figs. 7 and 8. In Fig. 8, we present the membrane ying-yang depolarization mediated by 20-HETE and PKC (31) (depolarizing action) and the opposing hyperpolarizing action mediated by EETs (9, 14, 17). Figure 7 depicts the response and interaction of the entire neurovascular unit comprising neurons, astrocytes, and the microvasculature. Signals triggered by the metabolic demand of activated neurons will initiate a sequence of events equivalent to the recordings of brain activity by functional magnetic resonance imaging in which astrocyte-derived dilators such as EETs increase blood oxygenation and blood flow to match metabolic demand (20, 24, 49). Such an occurrence of the sequence of events has been described in detail almost simultaneously by several laboratories, including ours (20, 24, 49).

Fig. 7.

Pressure and/or stretch activate PLC to release diacylglycerol (DAG) and AA. The AA thus released can be metabolized by membrane-bound CYP 4A enzymes to 20-HETE. 20-HETE causes phosphorylation of ion channels by activating PKC, in the case of K_Ca reducing its activity, while activating and promoting Ca²⁺ entry thorough L-type Ca²⁺ channel. These ionic mechanisms depolarize and activate arterial muscle, which is maintained as long as the pressure or stretch stimulus remains. Brain astrocytes or endothelial cells generate EETs, which inhibit this pressure-induced vasoactive process by enhancing the activities of K_Ca channel current and reducing inward Ca²⁺ current thereby dilating arterioles. +, activation; −, inhibition; PIP₂, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; COX, cyclooxygenase; TXA₂, thromboxane A₂; VSMC, vascular smooth muscle cell.

Fig. 8.

Schematic diagram illustrating cascade of second messenger signaling events and ion conductance mechanisms contributing to the initiation and development of pressure-induced myogenic constriction that is modulated by EETs or 20-HETE. LI_Ca, L-type Ca²⁺ channel current; SOCC, stretch-operated cation channels; P (circled), phosphorylated state of the channel.

Angiogenesis

The role of EETs with respect to the induction of angiogenesis in the central nervous system was first reported in cocultures of astrocytes and cerebral microvascular endothelial cells (37). EETs released from astrocytes increased thymidine incorporation into endothelial cells and initiated the formation of tubelike capillary structures, which could be blocked by the CYP inhibitor 17-octadecynoic acid (37, 59). Angiogenesis related to EETs was mediated by the release of these CYP epoxygenase metabolites of AA from astrocytes and could also be seen using synthetic plugs of Matrigel impregnated with EETs (59). This angiogenic process appears to involve the tyrosine kinase pathway based on the observation that genistein, a tyrosine kinase inhibitor, markedly attenuates this process (59). Other laboratories using in vivo and in vitro methods have confirmed the role of EETs in angiogenesis (57, 58). Other pathways through which EETs induce angiogenesis include MEK/MAPK, inositol phosphoinositide-3 kinase (PI3K)-Akt, and sphignosine kinase-1 signaling pathways (57, 58). When cultured with astrocytes, cerebral capillary endothelial cells can form intact vascular circuits including arterioles, capillaries, and a blood brain barrier (Fig. 9) (59).

Fig. 9.

Changes in morphology of astrocytes and cerebral capillary endothelial cells when cocultured. A: formation of capillary-like structures (arrow) double labeled with acetylated LDL labeled with 1,1′-dioctadecyl-1,3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL; red) and glial fibrillary acidic protein (GFAP; green) in the coculture of astrocytes and cerebral capillary endothelial cells. B: formation of capillary-like structures (arrow) triple labeled by platelet endothelial cell adhesion molecule-1 (PECAM-1; green), GFAP (red), and 4,6-diamidino-2-phenylindole (DAPI; blue) in the coculture of astrocytes and cerebral capillary endothelial cells. Note the interaction between astrocytes and endothelial tubes (arrowheads in A and B). C: double immunolabeling of blood vessels and astrocytes with PECAM-1 (green) and GFAP (red) in a section from normal rat cortex showing astrocytes from foot processes that impinge on vessels (arrows). D: cells (*) forming tube identified by DAPI staining (E) in coculture exhibit punctate staining of connexin-43 on their cell-cell borders (arrow). Arrow points to area shown in the inset at higher magnification. F: cells (*) outside tubes in the same coculture as in D show moderate to light cytoplasmic staining of connexin-43. Bar in A = 25 μm for A and B, bar = 20 μm in C, bar in F =10 μm for D–F, and bar in inset of D = 5 μm (59).

The Entire Myogenic Cascade in Cerebral Arterioles

One look at the cascade of signaling and second messenger molecules identified thus far makes it clear why we have been working on the mechanisms responsible for the pressure-induced autoregulation of cerebral blood flow for over 100 years (3). The exponential rise in the generation of reagents and methods to define such pathways has allowed us to understand many of the mechanisms responsible in providing adequate blood flow to metabolically active neurons. Figure 10 depicts our current understanding relating the function of ion channels, regulation of membrane potential, and the myriad of signaling molecules that sense and transduce changes in blood pressure (5, 11, 13, 15, 19, 22, 25, 27–31, 39, 44, 48). We, in collaboration with the laboratories of Dr. Richard Roman (University of Mississippi) and Dr. Howard Jacobs and other geneticists at the Medical College of Wisconsin developed a rat model to define the physiological and molecular mechanisms of pressure-induced autoregulation of cerebral blood flow. Two of the genes we have defined as having relative importance in this regard code for adducin-3 (ADD-3) and dual-specificity protein phosphatase (DUSP)-5 genes.

Fig. 10.

Schematic diagram illustrating cascade of second messenger signaling events and ion conductance mechanisms that contribute to the initiation and development of pressure-induced myogenic constriction. PC, phosphatidylcholine; Lyso-PA, lyso-phosphatidic acid; pHSP27, phosphorylated heat shock protein 27; SR, sarcoplasmic reticulum; MLCK, myosin light chain kinase; GPCR, G protein-coupled receptor; GEF, guanine nucleotide exchange factor; PPase, protein phosphatase; L_Ca, L-type Ca²⁺ channel.

DUSP-5, a member of the DUSP gene family, targets and causes dephosphorylation of molecules such as activated MAPK, ERK, JNK, phosphatase and tensin homologues deleted on chromosome 10 and regulator of Akt (PTEN), and P38 (a form of mitogen-activated protein kinase) (33, 38), critical signaling molecules also known to be involved in pressure- or stretch-induced myogenic responses (33, 38, 55). The other gene of interest is the ADD-3 gene, which similar to the DUSP-5, is also expressed in brain tissue and cerebral vessels (unpublished findings), functions to promote the spectrin-actin binding, and controls the rate of actin polymerization by capping actin filaments at the plasma membrane (35, 36), thereby in a position to initiate myogenic response. Adducin function is Ca²⁺ and calmodulin dependent and contains the MARCKS domain (35, 36), which is a target for phosphorylation by a family of kinases including PKC, tyrosine kinase, and [Rho]-kinase, or similar molecules that are important in signaling pressure- or stretch-induced myogenic responses (13, 25, 29, 30, 38, 55). Despite the fact that very little is known about the functional roles of DUSP-5 and ADD-3 genes in vasoregulation, it is important that we study and unravel the potential roles of the ADD-3 and DUSP-5 genes in the development and maintenance of pressure-induced myogenic autoregulation in the cerebral circulation. As we learn more about these genes and other molecules associated with them, our knowledge of the mechanisms controlling the myogenic autoregulation of blood flow in normal and disease states will be enhanced.

Summary

Since the early work of Bayliss (3), we have known that the blood vessel wall is capable of adapting to changes in transmural pressure. In 1984, Harder (19) elucidated the fact that increasing transmural pressure across the vessel wall induces membrane depolarization by reducing outward K⁺ current and increasing inward Ca²⁺ current through L-type Ca²⁺ channels. As discussed above, numerous second messengers could participate in initiating the signaling events that are at the heart of regulating myogenic tone, adjusting diameter through feedback mechanisms to maintain flow constant and adequate to meet neuronal metabolic demands. This review covers The 2009 Wiggers Lecture, sponsored by the Cardiovascular Section of the American Physiological Society. We have not presented the bulk of what was said during the lecture because of its preliminary and proprietary nature. Dr. David R. Harder is deeply honored to be awarded to give this prestigious lecture. The American Physiological Society has a long and rich history and has become the epitome of the concept “encompass use of all disciplines and techniques to answer questions related to the physiological function of living organisms.”

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).