Elevations In Transmural Pressure Research Articles

Abstract MP05: Knockdown of Gamma-adducin Expression Impairs the Myogenic Response of the Cerebral and Renal Arterioles

We recently identified a region on chromosome 1 containing 15 genes that rescues the impaired myogenic response in the afferent arteriole (Af-art) and the development of renal injury in Fawn Hood Hypertensive (FHH) rats. We also found an inactivating K572Q mutation in the gamma-Adducin (Add3) gene in FHH rats in this region that may play a causal role. To test this hypothesis, the present study examed the effect of knockdown of the expression of Add3 with DsiRNA on the myogenic response of renal and cerebral arterioles. A newly designed 27-mer Add3 DsiRNA blocked the expression of the Add3 protein in 293 cells and reduced Add3 mRNA expression in a dose-dependent manner up to 75% in cultured middle cerebral arteries (MCA). The inner diameter of MCA and renal Af-art 36 hours after co-transfection of Add3 DsiRNA dilated by 9 ± 2% and 4 ± 3%, respectively, in response to an elevation in transmural pressure from 60 to 140 mmHg. In contrast, these vessels still constricted normally by 11± 2% and 10 ± 1% in vessels transfected with scrambled siRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to NE (10-7 M). The large conductance calcium sensitive potassium (BK) current was 5-fold higher in vascular smooth muscle cells (VSMC) freshly isolated from the MCA that were transfected with Add3 DsiRNA as indicated by the appearance of SiGLO red fluorescence compared with that seen in non-transfected cells. Administration of IBTX normalized the elevated BK channel current recorded from VSMC transfected with Add3 DsiRNA, but it had little effect in non-transfected cells. These results indicate that knockdown of the expression of Add3 impairs the myogenic response of both MCA and Af-art and it is associated with an elevation in BK channel activity. It also supports the hypothesis that the K572Q mutation of Add3 found in FHH rats may play a causal role in the impaired myogenic response and autoregulation of renal and cerebral blood flow by elevating BK channel activity in VSMC.

Effects of Acute Transmural Pressure Elevation on Endothelium-Dependent Vasodilation in Isolated Rat Mesenteric Veins

Background/Aims: The vascular regulatory function of the endothelium can be impaired by increases in transmural pressure (TMP). We tested the hypothesis that increasing TMP impairs the endothelial dilator function of rat mesenteric small veins (MSVs). Methods: In PGF2α-preconstricted MSVs, bradykinin (BK), sodium nitroprusside (SNP) and S-Nitroso-N-acetylpenicillamine (SNAP) concentration-response curves were generated at intermediate (6 mm Hg) and high (12 mm Hg) pressures. BK-induced vasodilation was examined in the absence and presence of nitric oxide synthase inhibitor [N^ω-nitro-<smlcap>L</smlcap>-arginine (<smlcap>L</smlcap>-NNA), 100 µ<smlcap>M</smlcap>], cyclooxygenase inhibitor (indomethacin, 1 µ<smlcap>M</smlcap>), and large (BK_Ca, paxilline, 500 n<smlcap>M</smlcap>) and small (SK_Ca, apamin, 300 n<smlcap>M</smlcap>) conductance Ca²⁺-activated K⁺ channel blockers. Results: BK, SNP and SNAP responses were not altered by TMP increases. BK-induced vasodilation was significantly reduced by <smlcap>L</smlcap>-NNA, indomethacin, apamin and paxilline at 6 mm Hg and <smlcap>L</smlcap>-NNA at 12 mm Hg, and was further reduced by coapplication of apamin and/or paxilline with <smlcap>L</smlcap>-NNA compared with responses obtained with either blocker. Endothelium removal completely abolished BK-induced vasodilation. Conclusion: Venous endothelial dilator function is not affected by TMP elevation. BK-induced vasodilation is completely dependent on the presence of functional endothelial cells and mediated in part by nitric oxide, BK_Ca and SK_Ca channels, while the participation of prostacyclin may be important at intermediate pressures.

Effects of Etonogestrel Treatment in the Reproductive Organs and Uterine Arteries of Nonoophorectomized Guinea Pigs

The endometria of women treated with long-term progestin-only contraceptives (LTPOCs) display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow, oxidative stress, and unpredictable focal abnormal endometrial bleeding. Because human studies on the effects of LTPOC treatment are constrained for ethical and practical reasons, we assessed the suitability of nonoophorectomized guinea pigs (GPs) to best mimic the hormonal milieu of women. The present study demonstrates that treatment of GPs parallels the morphological changes following LTPOC treatment of the human endometrium and ovaries. Specifically, treatment resulted in larger hyperemic, uteri compared with controls. Histopathologic and immunohistochemical analysis demonstrated fewer endometrial glands, decreased luminal mucus, increased numbers of blood vessels, and focal hemorrhage. While increased staining for the cell mitosis marker, Ki67, was present in the zona functionalis, no such increase occurred in the basalis. Lastly, effects on vasomotor features of uterine arteries suggest changes that favor increased resistance and reduced blood flow promoting decreased ability to withstand elevations in transmural pressure.

TNFα is central to the augmented myogenic response of skeletal muscle resistance arteries in heart failure

A hallmark of heart failure (HF) is elevated peripheral resistance, which primarily results from an augmented myogenic response (MR, the intrinsic property of resistance arteries (RA) to adapt diameter to changes in pressure). In cerebral arteries from HF mice, tumor necrosis factor α (TNFα) plays a central role for the enhancement of MRs (through activation of sphingosine kinase‐1). We hypothesized that TNFα is equally important for the augmentation of MRs in skeletal muscle RA and hence, increased peripheral resistance in HF. HF was induced in Male C57/BL6 mice through surgical ligation of the left anterior descending coronary artery. 6–8wks post‐ligation, cremaster muscle RA were isolated and cannulated on a pressure myograph. Elevations in transmural pressure (20–100mmHg in 20mmHg steps) induced MRs that were increased in RA from HF compared to sham‐operated mice (n=7). MRs were not enhanced in RA from HF TNFα−/− mice (n=5). However, treatment with the TNFα scavenger etanercept (1mg/kg 2x/wk for 6wks post‐ligation) did not block the HF‐induced increase in MRs.Our data suggest that: (i) the augmented MR in skeletal muscle arteries contributes to increased vascular resistance in HF, (ii) TNFα is a mandatory component of this response, and (iii) chronic scavenging of TNFα might be compensated by redundant organization of MR signaling pathways.Funding: NSERC, HSFO, and CIHR

Tumor necrosis factor α is involved in the myogenic response of resistance arteries

The increase in vascular resistance observed in heart failure primarily results from an enhanced myogenic response (MR), the intrinsic property of resistance arteries (RA) to adapt their diameter and hence, resistance, to changes in pressure. We have recently shown that the cytokine tumor necrosis factor α (TNFα) augments the MR by activating sphingosine kinase‐1 (Sk1). We hypothesized that the TNFα‐Sk1 axis also operates under physiological conditions and that TNFα is an integral signaling element underlying the MR. Isolated hamster RA (n=6) were cultured in a pressure myograph for 20hrs prior to functional tests. Elevation of transmural pressure induced a robust MR (80±23% reversal of pressure‐induced distension). The soluble TNFα receptor etanercept (ETAN; 10μg/mL), which sequesters TNFα, abrogated the MR. This result was confirmed in mouse cremaster RA (n=5). Constriction to norepinephrine (NE) and dilation to acetylcholine (ACh) remained unaffected by ETAN. Heat‐inactivated ETAN (n=6) did not affect the MR or NE‐/ACh‐stimulated responses. TNFα increased Erk1/2 phosphorylation, which is tightly linked to Sk1 activation in resistance arteries.Our data suggest that: (i) TNFα is readily available in the artery wall, (ii) its activation is sensitive to changes in transmural pressure and (iii) TNFα is a key element of the signaling pathway that regulates the MR.Funding: NSERC/Heart and Stoke Foundation

Ca2+ sensitization via phosphorylation of myosin phosphatase targeting subunit at threonine‐855 by Rho kinase contributes to the arterial myogenic response

Ca(2+) sensitization has been postulated to contribute to the myogenic contraction of resistance arteries evoked by elevation of transmural pressure. However, the biochemical evidence of pressure-induced increases in phosphorylated myosin light chain phosphatase (MLCP) targeting subunit 1 (MYPT1) and/or 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) required to sustain this view is not currently available. Here, we determined whether Ca(2+) sensitization pathways involving Rho kinase (ROK)- and PKC-dependent phosphorylation of MYPT1 and CPI-17, respectively, contribute to the myogenic response of rat middle cerebral arteries. ROK inhibitors (Y27632, 0.03-10 micromol l(-1); H1152, 0.001-0.3 micromol l(-1)) and PKC inhibitors (GF109203X, 3 micromol l(-1); Gö6976; 10 micromol l(-1)) suppressed myogenic vasoconstriction between 40 and 120 mmHg. An improved, highly sensitive 3-step Western blot method was developed for detection and quantification of MYPT1 and CPI-17 phosphorylation. Increasing pressure from 10 to 60 or 100 mmHg significantly increased phosphorylation of MYPT1 at threonine-855 (T855) and myosin light chain (LC(20)). Phosphorylation of MYPT1 at threonine-697 (T697) and CPI-17 were not affected by pressure. Pressure-evoked elevations in MYPT1-T855 and LC(20) phosphorylation were reduced by H1152, but MYPT1-T697 phosphorylation was unaffected. Inhibition of PKC with GF109203X did not affect MYPT1 or LC(20) phosphorylation at 100 mmHg. Our findings provide the first direct, biochemical evidence that a Ca(2+) sensitization pathway involving ROK-dependent phosphorylation of MYPT1 at T855 (but not T697) and subsequent augmentation of LC(20) phosphorylation contributes to myogenic control of arterial diameter in the cerebral vasculature. In contrast, suppression of the myogenic response by PKC inhibitors cannot be attributed to block of Ca(2+) sensitization mediated by CPI-17 or MYPT1 phosphorylation.

Evidence that 20-HETE contributes to the development of acute and delayed cerebral vasospasm

Recent studies have indicated that arachidonic acid (AA) is metabolized by the cytochrome P4504A (CYP4A) enzymes in cerebral arteries to produce 20-hydroxyeicosatetraenoic acid (20-HETE) and that this compound has effects on cerebral vascular tone that mimic those seen following subarachnoid hemorrhage (SAH). In this regard, 20-HETE is a potent constrictor of cerebral arteries that decreases the open state probability of Ca2+-activated K+ channels through activation of protein kinase C (PKC). It increases the sensitivity of the contractile apparatus to Ca2+ by activating PKC and rho kinase. The formation of 20-HETE is stimulated by angiotensin II (AII), endothelin, adenosine triphosphate (ATP) and serotonin, and inhibited by NO, CO and superoxide radicals. Inhibitors of the formation of 20-HETE block the myogenic response of cerebral arterioles to elevations in transmural pressure in vitro and autoregulation of cerebral blood flow (CBF) in vivo. 20-HETE also plays an important role in modulating the cerebral vascular responses to vasodilators (NO and CO) and vasoconstrictors (AII, endothelin, serotonin). Recent studies have indicated that the levels of 20-HETE in cerebrospinal fluid (CSF) increase in rats, dogs and human patients following SAH and that inhibitors of the synthesis of 20-HETE prevent the acute fall in CBF in rats and reverse delayed vasospasm in both dogs and rats. This review examines the evidence that an elevation in the production of 20-HETE contributes to the initial fall in CBF following SAH and the later development of delayed vasospasm.

The Microvascular Effects of Sphingosine Kinase 1 are Regulated by its Subcellular Localization

Sphingosine kinase 1 (Sk1) is an important regulator of myogenic tone and smooth muscle cell Ca2+ sensitivity. Phosphorylation of Sk1 at serine 225, which results in its translocation to the plasma membrane, is required for full enzymatic activation. To investigate the functional role of Sk1 phosphorylation and localization, isolated resistance arteries from the hamster gacilis muscle were transfected with GFP or various mutants of Sk1. Elevation of transmural pressure stimulated the translocation of Sk1, but not a non-phosphorylatable mutant of Sk1 (SkS225A). Compared to GFP controls, expression of SkS225A reduced resting and myogenic tone, resting Ca2+, pressure-induced Ca2+ elevations and Ca2+ sensitivity. Inhibition of ERK1/2 signalling (PD98059), which putatively phosphorylates Sk1, had similar effects on resting and myogenic tone. In an attempt to rescue the effects of the S225A mutation, we employed a membrane anchored version of the mutant. Surprisingly, only resting Ca2+ and pressure-induced Ca2+ elevations were restored. Interestingly, expression of a membrane anchored wild-type Sk1 enzyme also reduced resting tone, myogenic vasoconstriction and Ca2+ sensitivity compared to GFP controls, but to a lesser extent than the anchored S225A mutant. We conclude that phosphorylation-dependent translocation of Sk1 to the plasma membrane is critical for its regulatory function in vascular smooth muscle cells. This process can not be imitated by forced localization of Sk1 with a membrane anchor.

Functional alterations in cerebrovascular K+ and Ca2+ channels are comparable between simulated microgravity rat and SHR

Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca(2+)-activated K(+) (BK(Ca)), voltage-gated K(+) (K(V)), and L-type voltage-dependent Ca(2+) (Ca(L)) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, K(V), BK(Ca), and Ca(L) currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. K(V) current density decreased in Sus rats vs. Con rats [1.07 +/- 0.14 (n = 22) vs. 1.31 +/- 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 1.70 +/- 0.37 (n = 23) vs. 0.88 +/- 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); Ca(L): -2.17 +/- 0.21 (n = 35) vs. -1.31 +/- 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: K(V) current density decreased [1.03 +/- 0.33 (n = 9) vs. 1.62 +/- 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 2.54 +/- 0.47 (n = 11) vs. 1.12 +/- 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); Ca(L): -3.99 +/- 0.53 (n = 12) vs. -2.28 +/- 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.

Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction.

Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The alpha1-adrenergic agonist phenylephrine (0.1-1 micromol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10-60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 micromol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 micromol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.

Interaction of myogenic mechanisms and hypoxic dilation in rat middle cerebral arteries.

The goal of this study was to determine how myogenic responses and vascular responses to reduced Po(2) interact to determine vascular smooth muscle (VSM) transmembrane potential and active tone in isolated middle cerebral arteries from Sprague-Dawley rats. Stepwise elevation of transmural pressure led to depolarization of the VSM cells and myogenic constriction, and reduction of the O(2) concentration of the perfusion and superfusion reservoirs from 21% O(2) to 0% O(2) caused vasodilation and VSM hyperpolarization. Myogenic constriction and VSM depolarization in response to transmural pressure elevation still occurred at reduced Po(2). Arterial dilation in response to reduced Po(2) was not impaired by pressure elevation but was significantly reduced at the lowest transmural pressure (60 mmHg). However, the magnitude of VSM hyperpolarization was unaffected by transmural pressure elevation. This study demonstrates that myogenic activation in response to transmural pressure elevation does not override hypoxic relaxation of middle cerebral arteries and that myogenic responses and hypoxic relaxation can independently regulate vessel diameter despite substantial changes in the other variable.

Redox signaling of the arteriolar myogenic response.

Arteriolar vascular smooth muscle cells (VSMCs) are mechanosensitive, constricting to elevations in transmural pressure (P(TM)). The goal of the present study was to determine using mouse isolated tail arterioles and arteries whether oxidant signaling regulates this myogenic response. In response to P(TM) elevation, VSMCs of arterioles but not arteries generated constriction and increased reactive oxygen species (ROS) activity (using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein). Arterioles had increased expression of NADPH oxidase components compared with arteries. Inhibition of NADPH oxidase, using mice with targeted impairment of enzyme components (p47(phox) or rac1) or diphenyleneiodonium, prevented the pressure-induced generation of ROS. When ROS activity was inhibited, either by inhibiting NADPH oxidase or with N-acetylcysteine, the myogenic constriction was abolished. The myogenic constriction was also inhibited by catalase, which inactivates H(2)O(2), but was unaffected by a cell-permeant mimic of superoxide dismutase (MnTMPyP). alpha(1)-Adrenergic constriction was not associated with altered ROS activity and was not affected by inhibition of NADPH oxidase or ROS. Exogenous H(2)O(2) constricted VSMCs of arterioles but not arteries. Thus, NADPH oxidase and ROS, in particular H(2)O(2), contribute to the myogenic response of arteriolar VSMCs.

Renal and cardiovascular actions of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids.

1. Arachidonic acid (AA) is metabolized by cytochrome P450 (CYP)-dependent pathways to epoxyeicosatrienoic acids (EET) and 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney and the peripheral vasculature. 2. The present short review summarizes the renal and cardiovascular actions of these important mediators. 3. Epoxyeicosatrienoic acids are vasodilators produced by the endothelium that hyperpolarize vascular smooth muscle (VSM) cells by opening Ca2+-activated K+ (KCa) channels. 20-Hydroxyeicosatetraenoic acid is a vasoconstrictor that inhibits the opening of KCa channels in VSM cells. Cytochrome P450 4A inhibitors block the myogenic response of small arterioles to elevations in transmural pressure and autoregulation of renal and cerebral blood flow in vivo. Cytochrome P450 4A blockers also attenuate the vasoconstrictor response to elevations in tissue PO2, suggesting that this system may serve as a vascular oxygen sensor. Nitric oxide and carbon monoxide inhibit the formation of 20-HETE and a fall in 20-HETE levels contributes to the activation of KCa channels in VSM cells and the vasodilator response to these gaseous mediators. 20-Hydroxyeicosatetraenoic acid also mediates the inhibitory actions of peptide hormones on sodium transport in the kidney and the mitogenic effects of growth factors in VSM and mesangial cells. A deficiency in the renal production of 20-HETE is associated with the development of hypertension in Dahl salt-sensitive rats. 4. In summary, the available evidence indicates that CYP metabolites of AA play a central role in the regulation of renal, pulmonary and vascular function and that abnormalities in this system may contribute to the pathogenesis of cardiovascular diseases.

Production of 20-HETE and its role in autoregulation of cerebral blood flow.

In the brain, pressure-induced myogenic constriction of cerebral arteriolar muscle contributes to autoregulation of cerebral blood flow (CBF). This study examined the role of 20-HETE in autoregulation of CBF in anesthetized rats. The expression of P-450 4A protein and mRNA was localized in isolated cerebral arteriolar muscle of rat by immunocytochemistry and in situ hybridization. The results of reverse transcriptase-polymerase chain reaction studies revealed that rat cerebral microvessels express cytochrome P-450 4A1, 4A2, 4A3, and 4A8 isoforms, some of which catalyze the formation of 20-HETE from arachidonic acid. Cerebral arterial microsomes incubated with [(14)C]arachidonic acid produced 20-HETE. An elevation in transmural pressure from 20 to 140 mm Hg increased 20-HETE concentration by 6-fold in cerebral arteries as measured by gas chromatography/mass spectrometry. In vivo, inhibition of vascular 20-HETE formation with N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS), or its vasoconstrictor actions using 15-HETE or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), attenuated autoregulation of CBF to elevations of arterial pressure. In vitro application of DDMS, 15-HETE, or 20-HEDE eliminated pressure-induced constriction of rat middle cerebral arteries, and 20-HEDE and 15-HETE blocked the vasoconstriction action of 20-HETE. Taken together, these data suggest an important role for 20-HETE in the autoregulation of CBF.

Altered renal hemodynamics and impaired myogenic responses in the fawn-hooded rat.

The present study examined whether an abnormality in the myogenic response of renal arterioles that impairs autoregulation of renal blood flow (RBF) and glomerular capillary pressure (PGC) contributes to the development of renal damage in fawn-hooded hypertensive (FHH) rats. Autoregulation of whole kidney, cortical, and medullary blood flow and PGC were compared in young (12 wk old) FHH and fawn-hooded low blood pressure (FHL) rats in volume-replete and volume-expanded conditions. Baseline RBF, cortical and medullary blood flow, and PGC were significantly greater in FHH than in FHL rats. Autoregulation of renal and cortical blood flow was significantly impaired in FHH rats compared with results obtained in FHL rats. Myogenically mediated autoregulation of PGC was significantly greater in FHL than in FHH rats. PGC rose from 46 +/- 1 to 71 +/- 2 mmHg in response to an increase in renal perfusion pressure from 100 to 150 mmHg in FHH rats, whereas it only increased from 39 +/- 2 to 53 +/- 1 mmHg in FHL rats. Isolated perfused renal interlobular arteries from FHL rats constricted by 10% in response to elevations in transmural pressure from 70 to 120 mmHg. In contrast, the diameter of vessels from FHH rats increased by 15%. These results indicate that the myogenic response of small renal arteries is altered in FHH rats, and this contributes to an impaired autoregulation of renal blood flow and elevations in PGC in this strain.

Possible role of nitric oxide in autoregulatory response in rat intracerebral arterioles.

Cerebral autoregulation is an important regulatory mechanism that maintains a constant cerebral blood flow over a wide range of perfusion pressures. The goal of this study was to determine whether nitric oxide contributes to the autoregulatory response of cerebral arterioles to altered transmural pressure (TMP). Seventy-nine intraparenchymal arterioles (53.6 +/- 3.5 microm mean diameter) isolated from rats were cannulated with micropipettes and pressurized at a TMP of 60 mm Hg (control pressure). Vessel diameters were monitored continuously using a video dimensional analyzer. The autoregulatory diameter responses to varying intraluminal pressures were observed in the presence and absence of a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA). The effect of L-NMMA-induced constriction on autoregulatory response also was compared with responses after prostaglandin F2alpha and alkalosis-induced constrictions. Autoregulatory responses were observed over a range from 10 to 90 mm Hg of TMP. Treatment with 10(-4) mol/L L-NMMA constricted arterioles and inhibited the autoregulatory vasodilation to TMP reductions from 60 mm Hg to 10 or 30 mm Hg. In L-NMMA-treated arterioles, elevation in TMP from 60 to 90 mm Hg caused an autoregulatory vasoconstriction. Treatment with alkaline pH 7.65 constricted arterioles to a similar degree as that induced by L-NMMA at 60 mm Hg, and under these conditions, the autoregulatory response remained intact. Arterioles severely constricted with prostaglandin F2alpha showed no significant autoregulatory response. These results suggest that 1) vascular nitric oxide release increases in response to a decrease in TMP from 60 mm Hg, thereby contributing to the autoregulatory vasodilation intrinsic to the vessel during hypotension, 2) arteriolar nitric oxide appears not to be involved in the autoregulatory vasoconstriction induced by elevating TMP from 60 to 90 mm Hg, and 3) a marked increase in vascular tone may affect autoregulatory response.

KCa-channel blockade prevents sustained pressure-induced depolarization in rat mesenteric small arteries.

In small blood vessels, elevation of transmural pressure induces myogenic constrictions and smooth muscle depolarization. The role of calcium-activated K+ channels (KCa channels) in these responses was examined in cannulated rat mesenteric small arteries. Inner and outer diameter were continuously monitored with a video technique. Smooth muscle membrane potential was recorded simultaneously using microelectrodes. To test for myogenic responsiveness, the transmural pressure was changed stepwise in the range between 10 and 120 mmHg. Pressure elevation induced moderate myogenic responses and significant depolarization, from -54.5 +/- 0.4 (SE) mV (n = 56) at 10 mmHg to -47.3 +/- 1.8 mV (n = 12) at 120 mmHg. Norepinephrine (NE, 0.67 and 10 microM) induced constriction and vasomotion, augmented myogenic responsiveness, and shifted the pressure-membrane potential relation to more depolarized values. Blockade of the Kca channels with charybdotoxin (ChTX) suppressed the responsiveness to pressure. In the absence of ChTX, with 0.67 microM NE, pressure elevation from 10 to 120 mmHg induced depolarization from -46.9 +/- 1.0 (n = 16) to -35.8 +/- 0.7 (n = 12) mV, whereas because of the myogenic response, the diameter increased only by 7%. In the presence of ChTX, with 0.3 microM NE, pressure changed the membrane potential from -41.0 +/- 1.1 (n = 12) to -37.8 +/- 0.7 mV (n = 4), which was not significant, and the diameter increased by 28%. These results demonstrate that blockade of KCa channels reduces responsiveness to pressure elevation. This suggests that pressure may induce depolarization and concomitant myogenic responsiveness by closure of KCa channels.

Regulation of membrane potential and diameter by voltage-dependent K+ channels in rabbit myogenic cerebral arteries.

The hypothesis that voltage-dependent K+ channels are involved in the regulation of arterial smooth muscle membrane potential and blood vessel diameter was tested by examining the effects of inhibitors [4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP)] of voltage-dependent K+ channels on the membrane potential and diameter of pressurized small (100- to 300-microns diam) cerebral arteries from rabbit. In response to graded elevations in transmural pressure (20-100 mmHg), the membrane potential of smooth muscle cells in these arteries depolarized and the arteries constricted. 4-AP (1 mM) and 3,4-DAP (1 mM) depolarized cerebral arteries by 19 and 21 mV, respectively, when they were subjected to a transmural pressure of 80 mmHg. 3-Aminopyridine (3-AP, 1 mM), which is a relatively poor inhibitor of voltage-dependent K+ channels, depolarized smooth muscle cells in the arteries by 1 mV. 4-AP and 3,4-DAP constricted pressurized (to 80 mmHg) cerebral arteries. 3-AP had little effect on arterial diameter. 4-AP increased the arterial constriction to transmural pressure over a wide range of pressures (40-90 mmHg). The effects of 4-AP and 3,4-DAP on membrane potential and diameter were not prevented by inhibitors of calcium channels, calcium-activated K+ channels, ATP-sensitive K+ channels, inward rectifier K+ channels, blockers of adrenergic, serotonergic, muscarinic, and histaminergic receptors, or removal of the endothelium. These results suggest that voltage-dependent K+ channels are involved in the regulation of membrane potential and response of small cerebral arteries to changes in intravascular pressure.

Transduction of physical force by the vascular wall: Role of phospholipase C and cytochrome P450 metabolites of arachidonic acid

The blood vessel wall responds actively to an elevation in transmural pressure. This pressure-induced myogenic response is thought to set the basal level of vascular tone upon which metabolic and neural influences operate in concert to regulate organ blood flow. The cellular mechanisms that mediate the vascular muscle response to mechanical deformation via a changing transmural pressure include membrane depolarization, activation of phospholipase C, and a rise in intracellular [Ca 2+] i, which appear to be nonadapting—remaining active as long as the pressure stimulus is applied. This brief review addresses some of the cellular events mediating transduction of transmural pressure by the vessel wall. Two possible mechanisms that are responsible for the nonadapting nature of pressure-induced myogenic tone are also explored, namely, formation of a P450 metabolite of arachidonic acid, which acts to buffer activation of K + channels as intracellular Ca 2+ rises, and direct activation of Ca 2+ channels by diacylglycerol. Evidence is provided suggesting that activation of phospholipase C is responsible for both the release of the arachidonic acid substrate for P450 enzymes and for the formation of diacylglycerol via its action on membrane-bound phospholipids.

Pressurization of isolated renal arteries increases inositol trisphosphate and diacylglycerol.

Inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) concentrations were measured in isolated, cannulated dog renal arteries under control conditions (0 mmHg) and in response to step elevations in transmural pressure. There was a pressure-dependent increase in IP3 at 60 and 120 mmHg, reaching significance at 120 mmHg (P < 0.05) and a significant increase in DAG at both 60 and 120 mmHg measured after maintaining pressure for 15 min. Similarly, IP3 measurements made 90 s after a step increase in transmural pressure also exhibited a pressure-dependent profile, again reaching significance at 120 mmHg. Calculation of active tension demonstrated these renal arteries developed pressure-dependent myogenic tone. To assess the role of the endothelium in this regard, IP3 was measured before and after endothelial removal at 0 and 60 mmHg. Pressure-dependent myogenic tone was still present upon endothelial removal. In the absence of the endothelium, we observed a significant increase in total IP3 at 60 compared with 0 mmHg; furthermore, the increase in IP3 in the absence of the endothelium was significantly greater than that observed when the endothelium was intact. Given that the primary source of IP3 is via the actions of phospholipase C (PLC) on phosphatidylinositol 4,5-bisphosphate, these biochemical data directly demonstrate that elevation of transmural pressure in dog renal arteries activates PLC.