Regulation of arterial tone by smooth muscle myosin type II

Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Charité University Hospital, Humboldt University of Berlin, 13125 Berlin; and Medical School Hannover, Department of Nephrology, D-30625 Hannover, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The initiation of contractile force in arterial smooth muscle (SM) is believed to be regulated by the intracellular Ca²⁺ concentration and SM myosin type II phosphorylation. We tested the hypothesis that SM myosin type II operates as a molecular motor protein in electromechanical, but not in protein kinase C (PKC)-induced, contraction of small resistance-sized cerebral arteries. We utilized a SM type II myosin heavy chain (MHC) knockout mouse model and measured arterial wall Ca²⁺ concentration ([Ca²⁺]_i) and the diameter of pressurized cerebral arteries (30-100 µm) by means of digital fluorescence video imaging. Intravasal pressure elevation caused a graded [Ca²⁺]_i increase and constricted cerebral arteries of neonatal wild-type mice by 20-30%. In contrast, intravasal pressure elevation caused a graded increase of [Ca²⁺]_i without constriction in (/) MHC-deficient arteries. KCl (60 mM) induced a further [Ca²⁺]_i increase but failed to induce vasoconstriction of (/) MHC-deficient cerebral arteries. Activation of PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM) induced a strong, sustained constriction of (/) MHC-deficient cerebral arteries without changing [Ca²⁺]_i. These results demonstrate a major role for SM type II myosin in the development of myogenic tone and Ca²⁺-dependent constriction of resistance-sized cerebral arteries. In contrast, the sustained contractile response did not depend on myosin and intracellular Ca²⁺ but instead depended on PKC. We suggest that SM myosin type II operates as a molecular motor protein in the development of myogenic tone but not in pharmacomechanical coupling by PKC in cerebral arteries. Thus PKC-dependent phosphorylation of cytoskeletal proteins may be responsible for sustained contraction in vascular SM.

protein kinase C; pressurized cerebral arteries; phorbol ester; arterial tone; myosin heavy chain; knockout mouse

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CONTRACTION OF SMOOTH MUSCLE (SM) is based on a sliding-filament mechanism similar to that of striated muscle. The SM contraction is regulated by electromechanical as well as by pharmacomechanical coupling mechanisms. Excitation-contraction coupling by electromechanical mechanisms depends on the membrane potential and Ca²⁺ influx through voltage-gated Ca²⁺ channels in the plasma membrane, such as L-type Ca²⁺ channels (38). In small arteries, membrane depolarization from 50 to 40 mV increases external Ca²⁺ influx, leading to an increase in global intracellular Ca²⁺ concentration ([Ca²⁺]_i) and subsequent activation of the contractile machinery. Lowering [Ca²⁺]_i reverses contraction (17). Conclusively, force produced by electromechanical coupling depends on Ca²⁺ ions surrounding the contractile filaments. In contrast, Ca²⁺ ions are believed to play a minor role in pharmacomechanical coupling. The activation of protein kinase C (PKC) and other intracellular enzymes seems to play a key role in force production by many vasoconstrictor hormones (38).

SM contraction exhibits several distinct properties that are not present in skeletal muscle. For example, the regulation of crossbridges interaction in SM contraction by Ca²⁺ seems to be more complicated than in striated muscle (32). Accordingly, pharmacomechanical coupling of SM can be induced in Ca²⁺-free external solutions, suggesting additional, Ca²⁺-independent pathways of SM contraction (8). We used the SM myosin type II heavy chain (MHC) knockout mouse to provide evidence that SM myosin type II operates as the molecular motor protein for depolarization-induced, Ca²⁺-dependent constriction of small cerebral arteries, i.e., in electromechanical coupling. In addition, we show that PKC-dependent, sustained constriction does not depend on intracellular Ca²⁺ and SM myosin type II. We suggest that this myosin-independent contractile pathway may play a major role in pharmacomechanical coupling of small, resistance-sized arteries.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cerebral arteries. Cerebral arteries were obtained from neonatal (+/+) and (/) SM-MHC-deficient mice genotyped after the experiment (30). After decapitation, the brain was removed and quickly transferred to cold (4°C), oxygenated (95% O₂/5% CO₂) physiological salt solution (PSS) of the following composition (in mM): 119 NaCl, 4.7 KCl, 25 NaHCO₃, 1.2 KH₂PO₄, 1.6 CaCl₂, 1.2 MgSO₄, 0.03 EDTA, and 11 glucose. Thereafter, the cerebral arteries were dissected from the brain and placed in cold PSS. Connective tissue was removed. For measurements of intracellular arterial wall [Ca²⁺]_i, the vessels were incubated with the Ca²⁺-sensitive indicator fura 2-AM (5 µM) and pluronic acid (0.005%; wt/vol) for 45 min at room temperature in oxygenated PSS. After loading with fura 2-AM, the arteries were cannulated in a chamber on glass canulas on both sides, allowing an application of hydrostatic pressure to the vessel (for details, see Refs. 6, 9, 26, 29). [Ca²⁺]_i and diameter were measured simultaneously by using a conventional spectrometer (dm3000; SPEX, Edison, NJ) and a videomicroscopic system (Nikon Diaphot, Düsseldorf, Germany) connected to a personal computer with appropriate software for detection of changes of vessel diameter (TSE, Bad Homburg, Germany) (for calibration and details, see Ref. 9). All experiments were performed at 37°C.

Materials and statistics. Fura 2-AM was purchased from Molecular Probes (Eugene, OR). Stock solutions (0.25 mM) of fura 2-AM were made using DMSO as the solvent. All salts and drugs were obtained from Sigma Aldrich (Deisenhofen, Germany) or Merck (Darmstadt, Germany). High external potassium solutions were made by iso-osmotic substitution of NaCl with KCl in the PSS. All values are given as means ± SE. For group comparisons, paired and unpaired Student' s t-tests or nonparametric Wilcoxon tests were used as appropriate. A value of P < 0.05 was considered statistically significant; n = number of arteries tested.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Electromechanical coupling. We first measured intracellular arterial wall [Ca²⁺]_i and vessel diameter of isolated cerebral arteries of (+/+) and (/) SM-MHC mice at an intravasal pressure of 10 mmHg. The cerebral arteries of (+/+) and (/) SM-MHC mice showed no macroscopic differences, for instance in diameter or branching. Figure 1 shows that resting intracellular [Ca²⁺]_i (~100 nM) and diameter (~80 µm) were not different (P < 0.05). In cerebral arteries of (+/+) SM-MHC mice, an increase in intravasal pressure from 30 to 60 mmHg induced an increase in [Ca²⁺]_i and decreased vessel diameter (myogenic constriction) by 20 µm (Figs. 2B and 4B; P < 0.05). To evaluate electromechanical coupling in cerebral arteries of (+/+) SM-MHC mice, high concentrations of external K⁺ (60 mM) were applied to the arteries and evoked an increase in [Ca²⁺]_i to ~1,000 nM and an additional 20% constriction in the vessels of (+/+) SM-MHC mice, compared with controls (Figs. 2B and 4B; P < 0.05).

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Comparison of resting intracellular arterial wall Ca2+ (A) and vessel diameter (B) in (/) myosin heavy chain (MHC)-deficient cerebral arteries and (+/+) controls. Parameters were not significantly different (n  8). Intravascular pressure was 10 mmHg.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Development of myogenic tone in (+/+) MHC wild-type cerebral arteries. A: schematic model of myogenic tone development. Increase in intravascular pressure induces membrane depolarization of arterial smooth muscle (SM) cells. Depolarization opens voltage-dependent Ca2+ channels, leading to an increase in global intracellular Ca2+ concentration (9, 23, 26). The increase in global intracellular Ca2+ activates the contractile apparatus in the cell, leading to an actin-myosin II interaction and, subsequently, to cell contraction. B: simultaneous measurement of intracellular arterial wall Ca2+ and vessel diameter. Intravascular pressure was increased stepwise from 10 to 60 mmHg. The increase in intravascular pressure increased arterial wall Ca2+ (upper trace) and induced myogenic tone (lower trace, decrease of vessel diameter). Application of 60 mM KCl evoked an additional increase in arterial wall Ca2+ and induced vasoconstriction. The presence of KCl is indicated by the horizontal line.

In contrast, cerebral arteries of (/) SM-MHC mice did not develop myogenic constriction when intravascular pressure was increased to 30 or to 60 mmHg (Figs. 3B and 4A). Instead, the vessel diameter was "passively" increased by these maneuvers by 15 to 20% (Fig. 4A; P < 0.05). However, the stepwise increase in intravascular pressure from 10 to 60 mmHg induced a graded increase in [Ca²⁺]_i up to 200 nM (P < 0.05). High concentrations of external K⁺ (60 mM) were applied to the arteries of (/) SM-MHC mice to test whether or not the electromechanical coupling in these arteries was functional. Application of 60 mM KCl to arteries of (/) SM-MHC mice evoked an increase in [Ca²⁺]_i to ~1,000 nM (Figs. 3B and 4A; P < 0.05). However, this maneuver did not constrict the vessels (Figs. 3B and 4A; P < 0.05). These findings indicate that pressure-induced and membrane potential-dependent Ca²⁺ influx mechanisms are intact in arteries of (/) SM-MHC mice (Fig. 3B). However, in sharp contrast to arteries of (+/+) SM-MHC mice, Ca²⁺-dependent, electromechanical coupling was not functional in cerebral arteries lacking SM-MHC (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Lack of myogenic tone and KCl induced vasoconstriction in (/) MHC-deficient cerebral arteries. A: proposed schematic model of absent myogenic tone. B: simultaneous measurement of arterial wall Ca2+ and vessel diameter. Intravascular pressure was increased stepwise from 10 to 60 mmHg. This increase induced a sustained increase in arterial wall Ca2+ (upper trace). However, this increase in Ca2+ did not cause myogenic constriction of the artery (lower trace). Subsequent application of KCl (60 mM) increased arterial wall Ca2+ but did not cause vasoconstriction.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Changes in arterial wall Ca2+ and vessel diameter of (/) MHC cerebral arteries and (+/+) controls induced by intravascular pressure and 60 mM KCl. Changes are expressed as a percentage of steady-state change in arterial wall [Ca2+]i (arbitrary units in %; 100%) and steady-state vessel diameter (100%) obtained with 10 mmHg before administration of 30 mmHg, 60 mmHg, or 60 mM KCl (n  12). A: arteries of (/) SM-MHC mice did not develop the myogenic response or constricted to 60 mM KCl. B: arteries of (+/+) SM-MHC mice showed a reduced diameter in response to increased intravascular tone or KCl.

PKC-induced constriction. We next tested the hypothesis that PKC induces pharmacomechanical coupling in cerebral arteries. In the presence of external Ca²⁺, the PKC activator phorbol 12-myristate 13-acetate (PMA, 100-500 nM) induced a strong constriction of arteries of (+/+) SM-MHC mice (Figs. 5B and 7B; P < 0.05). PMA did not increase [Ca²⁺]_i (Figs. 5B and 7B; P > 0.05). The SM constriction induced by PMA was rapid and reached the maximum (~50-60% constriction) after 2 min (half-activation time of contraction ~99 s; Fig. 5B). This PMA effect was not attenuated in both nominal Ca²⁺-free and Ca²⁺-buffered [5 mM N-(2-hydrohyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA)] solutions (P > 0.05). These findings indicate that PKC activation induces a fast, sustained, and relatively Ca²⁺-independent induced constriction of cerebral arteries of (+/+) SM-MHC mice.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effects of phorbol 12-myristate 13-acetate (PMA) on arterial wall [Ca2+]i and diameter of (+/+) MHC, wild-type cerebral arteries. A: proposed schematic model of arterial tone regulation by actin-myosin II interaction and protein kinase C activation. B: application of external KCl (60 mM) evoked an increase in arterial wall Ca2+ (upper trace) and a strong constriction (lower trace). Subsequent application of PMA (500 nM) induced a rapid vasoconstriction within 2 min (half-activation time of contraction ~99 s). Arterial wall Ca2+ was only slightly increased. The constriction was maintained in nominal Ca2+-free solution (PSS without CaCl2) and even in strong Ca2+-buffered [nominal Ca2+-free solution plus 5 mM N-(2-hydrohyethyl)ethylenediamine-N,N',N'- triacetic acid (HEDTA)] solution. PMA, external Ca2+-free solutions, and KCl are indicated by horizontal lines.

In contrast, PMA (100-500 nM) evoked a delayed constriction of (/) MHC-deficient arteries by ~30% (half-activation time of contraction ~540 s; Figs. 6, A and C, and 7A; P < 0.05). PMA did not significantly change [Ca²⁺]_i (P > 0.05). Removal of external Ca²⁺ or strongly buffered Ca²⁺-free (5 mM HEDTA) external bath solution did not attenuate the PMA-induced vessel constriction (Figs. 6C and 7A; P > 0.05). Subsequent application of external KCl (60 mM) induced an increase in [Ca²⁺]_i to ~1 µM in cerebral arteries of (/) SM-MHC mice (P < 0.05) but did not induce further vessel constriction (P > 0.05). Cerebral artery constriction induced by stimulation of PMA was completely inhibited by a 10-min preincubation with calphostin C (100 nM; data not shown; n = 3).

View larger version (53K): [in this window] [in a new window]   Fig. 6.   Effects of PMA on arterial wall [Ca2+]i and diameter of (/) MHC-deficient cerebral arteries. A: image of a cannulated intact cerebral artery before and after stimulation of PKC by 500 nM PMA. B: schematic model of PMA-induced constriction of (/) MHC-deficient cerebral arteries. C: PMA (500 nM) induced a delayed (half-activation time of contraction ~540 s) and sustained constriction that was not influenced by removal of external Ca2+ or use of a strong, buffered, Ca2+-free external solution (HEDTA). Subsequent 60 mM KCl application in normal, Ca2+-containing PSS solution did not induce a further constriction but solely increased arterial wall Ca2+ concentration. PMA, external Ca2+-free solutions, and KCl are indicated by horizontal lines.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Changes of arterial wall [Ca2+]i and vessel diameter by PMA (100-500 nM) in (/) and (+/+) MHC-deficient cerebral arteries (n  8). Changes are expressed as a percentage changes of the steady-state in arterial wall [Ca2+]i (A: arbitrary units in %, 100%) and steady-state vessel diameter (B: 100%) obtained before administration of PMA (n  12). A: application of phorbol ester evoked a ~40% constriction of arteries of (/) SM-MHC mice. The phorbol ester evoked constriction was maintained in Ca2+-free or Ca2+-buffered bath solution (A). B: application of phorbol ester evoked in arteries of (+/+) SM-MHC mice a ~55% vasoconstriction. The phorbol ester induced constriction did not depend on extracellular calcium.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The mechanisms that lead to sustained SM contraction that occurs in arterial vasospasm and arterial hypertension remain unknown (39). We used the SM-MHC-deficient (/) mouse to characterize excitation-contraction coupling in myogenic cerebral arteries. Our data suggest that SM-MHC type II operates as a molecular motor protein in electromechanical coupling but not in pharmacomechanical coupling mediated by PKC of cerebral arteries. Electromechanical coupling in cerebral arteries of (+/+) and (/) SM-MHC mice was tested by elevating intravascular pressure to induce myogenic tone and by application of high potassium chloride (60 mM KCl) (1, 2, 13, 22, 28). Elevated intravasal pressure (60 mmHg) and application of 60 mM KCl increased [Ca²⁺]_i and reduced vessel diameter in cerebral arteries of (+/+) SM-MHC mice. In contrast, arteries of (/) SM-MHC mice did not develop myogenic tone but nevertheless showed normal Ca²⁺ transport regulation. Because of normal pressure- and K⁺-induced [Ca²⁺]_i increases in arteries of (/) SM-MHC mice, we assume that the initial step of electromechanical coupling in SM is functional in these arteries. However, the lack of SM contraction indicates that the contraction signal could not be converted into force by the contractile apparatus. The results indicate that other myosin isoforms could not assume force production in the absence of SM myosin type II. This finding is contrary to the suggestions by Morano et al. (30), who studied the urinary bladder of (/) SM-MHC mice (30). They reported that SM strips from urinary bladder of (/) MHC mice showed a sustained contraction after high doses of KCl (60 mM). We failed to observe a fast initial (phase 1) or a delayed (phase 2) contraction in cerebral artery preparations, as reported for urinary bladder (30). Morano et al. (30) suggested that nonmuscle myosin assumed the function of SM myosin type II to induce contraction in (/) SM myosin urinary bladder. However, because we did not observe contraction in (/) SM-MHC-deficient mice, it is possible that the complete myosin-actin-dependent contractile machinery is not functional in vascular SM of (/) SM-MHC-deficient cerebral arteries. Moreover, the data demonstrate that SM type II myosin operates a molecular motor protein in the development of myogenic tone and electromechanical coupling of cerebral arteries (Fig. 8).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Proposed model of electromechanical (A) vs. pharmacomechanical (B) coupling induced by protein kinase C in cerebral arteries. Electromechanical coupling depends on membrane potential and opening of voltage-gated Ca2+ channels. Ca2+ influx from the extracellular space and Ca2+ release from the internal stores activate the myosin-actin machinery and lead to contraction of the SM cell. Ca2+ and SM myosin type II play a major regulatory role in electromechanical coupling. In contrast, pharmacomechanical coupling induced by protein kinase C activation is relatively insensitive to changes of membrane potential and [Ca2+]i. We suggest that this pathway is mediated by protein kinase C-induced phosphorylation of an unknown intracellular motor protein. Possible candidates are a cytoskeletal protein or nonidentified myosin isoforms.

PKC-induced arterial constriction of MHC-deficient mice. Many hormonal vasoconstrictors activate PKC and, thereby, induce "pharmacomechanical" contraction of arterial SM (10). Analysis of PKC activation in SM revealed that during this process, some PKC isoforms translocate to the membrane (11, 12, 19, 20, 21, 24, 37) and are involved in the Ca²⁺ sensitization of the intact SM (3, 14, 25, 34). As shown by others, PKC activation induces a sustained arterial SM contraction in arteries expressing SM-MHC type II (3, 5, 16, 31, 36) with a slight (35) or no increase in [Ca²⁺]_i (15, 40).

We observed also that lowering extracellular Ca²⁺, by using nominal Ca²⁺-free bath solution or strong buffered Ca²⁺-free bath solution, did not influence PKC-induced contraction of wild-type cerebral arteries. Moreover, our results show that the PKC-induced vessel constriction is still inducible in arteries of (/) SM-MHC mice. Thus we suggest that PKC-induced contraction in cerebral arteries of (+/+) SM-MHC mice may involve two components. The first is a SM myosin type II-mediated contraction with a fast onset, within 2 min after PKC stimulation. The second is a contraction mediated by a molecular motor protein other than SM myosin type II, namely a non-SM-myosin type II motor protein (possibly nonmuscle myosin). The latter molecular motor protein is turned on by PKC in cerebral arteries with a slow onset, ~15 min after PKC stimulation. According to this model, we observed only the delayed non-SM myosin type II motor protein-mediated constriction with a slow onset (15 min) in arteries of (/) SM-MHC mice. Extracellular Ca²⁺ is not necessary to maintain this contractile mechanism.

In this context, it is noteworthy that PKC activation leads to changes in the cell cytoskeleton in a variety of cells and may represent an important regulator of cytoskeletal functions (18). Platts et al. (33) showed that the microtubule network is involved in arterial SM contraction. Using pharmacological tools, they induced changes in the vascular SM cell microtubular network and observed constriction of the vessel without changes in [Ca²⁺]_i. These effects were not dependent on the endothelium (33). Thus PKC-dependent phosphorylation of cytoskeletal proteins may be responsible for sustained contraction in vascular SM, including those in cerebral arteries of (/) SM-MHC mice (Fig. 8). The cytoskeleton may interact with novel, still unidentified cytoplasmic proteins to produce contraction. Such proteins could serve as a substrate for PKC. They should be tightly associated with the cytoskeleton, and binding should be possibly Ca²⁺ independent. A short protein with these attributes was recently found in SM (4, 7, 27).

In conclusion, we provide indirect evidence for a novel molecular motor protein that is able to induce sustained, Ca²⁺-independent contraction of arterial SM. This nonmyosin type II motor protein can be activated by PKC and seems to play a major role in pharmacomechanical coupling of arterial SM. Further, SM myosin type II operates as a key element in the development of myogenic tone and electromechanical coupling of small, resistance-sized cerebral arteries. Pharmacomechanical coupling mediated by a nonmyosin type II is of interest in various pathophysiological conditions, including hypertension, coronary vasospasm, and migraine. It remains to characterize the molecular motor, and further studies are needed to clarify this point.

	ACKNOWLEDGEMENTS

We thank Drs. Ingo Morano and M. Bader, Max Delbrück Center Berlin, for providing the MHC-deficient mice.

	FOOTNOTES

This work was supported by the Deutsche Forschungsgemeinschaft.

Address for reprint requests and other correspondence: M. Gollasch, Franz Volhard Clinic, Wiltbergstrasse 50, 13125 Berlin, Germany (E-mail: gollasch{at}fvk-berlin.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 26, 2002;10.1152/ajpcell.01369.2000

Received 12 December 2000; accepted in final form 24 June 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1.   Bayliss, WM. On the local reactions of the arterial wall to changes of internal pressure. J Physiol 28: 220-231, 1902.

2.   Brayden, JE, and Nelson MT. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 256: 532-535, 1992[Abstract/Free Full Text].

3.   Chatterjee, M, and Tejada M. Phorbol ester-induced contraction in chemically skinned vascular smooth muscle. Am J Physiol Cell Physiol 251: C356-C361, 1986[Abstract/Free Full Text].

4.   Dammeier, S, Lovric J, Eulitz M, Kolch W, Mushinski JF, and Mischak H. Identification of the smooth muscle-specific protein sm22, as a novel protein kinase C substrate using two-dimensional gel electrophoresis and mass spectrometry. Electrophoresis 21: 2443-2453, 2000[ISI][Medline].

5.   Danthuluri, NR, and Deth RC. Phorbol ester-induced contraction of arterial smooth muscle and inhibition of -adrenergic response. Biochem Biophys Res Commun 125: 1103-1109, 1984[ISI][Medline].

6.   Duling, BR, Gore RW, Dacey RG, and Damon DN. Methods for isolation, cannulation, and in vitro study of single microvessels. Am J Physiol Heart Circ Physiol 260: H130-H135, 1991[Abstract/Free Full Text].

7.   Fu, Y, Liu HW, Forsythe SM, Kogut P, McConville JF, Halayko AJ, Camoretti-Mercado B, and Solway J. Mutagenesis analysis of human SM22: characterization of actin binding. J Appl Physiol 89: 1985-1990, 2000[Abstract/Free Full Text].

8.   Gokina, NI, and Osol G. Temperature and protein kinase C modulate myofilament Ca²⁺ sensitivity in pressurized rat cerebral arteries. Am J Physiol Heart Circ Physiol 274: H1920-H1927, 1998[Abstract/Free Full Text].

9.   Gollasch, M, Wellman GC, Knot HJ, Jaggar JH, Damon DH, Bonev AD, and Nelson MT. Ontogeny of local sarcoplasmic reticulum Ca²⁺ signals in cerebral arteries: Ca²⁺ sparks as elementary physiological events. Circ Res 83: 1104-1114, 1998[Abstract/Free Full Text].

10.   Griendling, KK, Tsuda T, and Alexander RW. Endothelin stimulates diacylglycerol accumulation and activates protein kinase C in cultured vascular smooth muscle cells. J Biol Chem 264: 8237-8240, 1989[Abstract/Free Full Text].

11.   Haller, H, Maasch C, Lindschau C, Brachmann M, Buchner K, and Luft FC. Intracellular targeting and protein kinase C in vascular smooth muscle cells: specific effects of different membrane-bound receptors. Acta Physiol Scand 164: 599-609, 1998[ISI][Medline].

12.   Haller, H, Smallwood JI, and Rasmussen H. Protein kinase C translocation in intact vascular smooth muscle strips. Biochem J 270: 375-381, 1990[ISI][Medline].

13.   Harder, DR. Pressure-dependent membrane depolarization in cat middle cerebral artery. Circ Res 55: 197-202, 1984[Abstract/Free Full Text].

14.   Jensen, PE, Gong MC, Somlyo AV, and Somlyo AP. Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca²⁺ sensitization of myosin light chain phosphorylation in smooth muscle. Biochem J 318: 469-475, 1996.

15.   Jiang, M, and Morgan KG. Intracellular calcium levels in phorbol ester-induced contractions of vascular muscle. Am J Physiol Heart Circ Physiol 253: H1365-H1371, 1987[Abstract/Free Full Text].

16.   Jiang, MJ, and Morgan KG. Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle. Pflügers Arch 413: 637-643, 1989[ISI][Medline].

17.   Kamm, KE, and Stull JT. Regulation of smooth muscle contractile elements by second messengers. Annu Rev Physiol 51: 299-313, 1989[ISI][Medline].

18.   Keenan, C, and Kelleher D. Protein kinase C and the cytoskeleton. Cell Signal 10: 225-232, 1998[ISI][Medline].

19.   Khalil, RA, Lajoie C, and Morgan KG. In situ determination of [Ca²⁺]_i threshold for translocation of the -protein kinase C isoform. Am J Physiol Cell Physiol 266: C1544-C1551, 1994[Abstract/Free Full Text].

20.   Khalil, RA, Lajoie C, Resnick MS, and Morgan KG. Ca²⁺-independent isoforms of protein kinase C differentially translocate in smooth muscle. Am J Physiol Cell Physiol 263: C714-C719, 1992[Abstract/Free Full Text].

21.   Khalil, RA, and Morgan KG. Phenylephrine-induced translocation of protein kinase C and shortening of two types of vascular cells of the ferret. J Physiol 455: 585-599, 1992[Abstract/Free Full Text].

22.   Knot, HJ, and Nelson MT. Regulation of membrane potential and diameter by voltage-dependent K-channels in rabbit myogenic cerebral arteries. Am J Physiol Heart Circ Physiol 269: H348-H355, 1995[Abstract/Free Full Text].

23.   Knot, HJ, and Nelson MT. Regulation of arterial diameter and wall [Ca²⁺] in cerebral arteries of rat by membrane potential and intravascular pressure. J Physiol 508: 199-209, 1998[Abstract/Free Full Text].

24.   Lee, TS, Chao T, Hu KQ, and King GL. Endothelin stimulates a sustained 1,2-diacylglycerol increase and protein kinase C activation in bovine aortic smooth muscle cells. Biochem Biophys Res Commun 162: 381-386, 1989[ISI][Medline].

25.   Lee, MW, and Severson DL. Signal transduction in vascular smooth muscle: diacylglycerol second messengers and PKC action. Am J Physiol Cell Physiol 267: C659-C678, 1994[Abstract/Free Full Text].

26.   Lohn, M, Gollasch M, Furstenau M, Morano I, Luft FC, and Haller H. Tonic vascular contraction is independent of myosin heavy chain and calcium (Abstract). Kidney Blood Press Res 22: 189, 1999.

27.   Mack, CP, Somlyo AV, Hautmann M, Somlyo AP, and Owens GK. Smooth muscle differentiation marker gene expression is regulated by rhoA-mediated actin polymerization. J Biol Chem 276: 341-347, 2000[Abstract/Free Full Text].

28.   Meininger, GA, and Davis MJ. Cellular mechanisms involved in the vascular myogenic response. Am J Physiol Heart Circ Physiol 263: H647-H659, 1992[Abstract/Free Full Text].

29.   Miller, FJ, Dellsperger KC, and Gutterman DD. Myogenic constriction of human coronary arterioles. Am J Physiol Heart Circ Physiol 273: H257-H264, 1997[Abstract/Free Full Text].

30.   Morano, I, Chai GX, Baltas LG, Lamounier-Zepter V, Lutsch G, Kott M, Haase H, and Bader M. Smooth-muscle contraction without smooth-muscle myosin. Nat Cell Biol 2: 371-375, 2000[ISI][Medline].

31.   Morgan, KG, and Leinweber BD. PKC-dependent signalling mechanisms in differentiated SM. Acta Physiol Scand 164: 485-505, 1998.

32.   Murphy, RA. Contraction in smooth muscle cells. Annu Rev Physiol 51: 275-283, 1989[ISI][Medline].

33.   Platts, SH, Falcone JC, Holton WT, Hill MA, and Meiniger GA. Alteration of microtubule polymerization modulates arteriolar vasomotor protein tone. Am J Physiol Heart Circ Physiol 277: H100-H106, 1999[Abstract/Free Full Text].

34.   Rasmussen, H, Kojima I, Kojima K, Zawalich W, and Apfeldorf W. Calcium as intracellular messenger: sensitivity modulation, C-kinase pathway, and sustained cellular response. Adv Cyclic Nucleotide Protein Phosphorylation Res 18: 159-193, 1984[ISI][Medline].

35.   Rembold, CM, and Murphy RA. [Ca²⁺]-dependent myosin phosphorylation in phorbol diester stimulated smooth muscle contraction. Am J Physiol Cell Physiol 255: C719-C723, 1988[Abstract/Free Full Text].

36.   Singer, HA, and Baker KM. Calcium dependence of phorbol 12,13-dibutyrate-induced force and myosin light chain phosphorylation in arterial smooth muscle. J Pharmacol Exp Ther 243: 814-821, 1987[Abstract/Free Full Text].

37.   Singer, HA, Schworer CM, Sweeley C, and Benscoter H. Activation of protein kinase C isozymes by contractile stimuli in arterial smooth muscle. Arch Biochem Biophys 299: 320-329, 1992[ISI][Medline].

38.   Somlyo, AP, and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994[Medline].

39.   Throckmorton, DC, Packer CS, and Brophy CM. Protein kinase C activation during Ca²⁺-independent vascular smooth muscle contraction. J Surg Res 78: 48-53, 1998[ISI][Medline].

40.   Walsh, MP, Andrea JE, Allen BG, Clement-Chomienne O, Collins EM, and Morgan KG. Smooth muscle protein kinase C. Can J Physiol Pharmacol 72: 1392-1399, 1994[ISI][Medline].