-adrenoceptor activation of arteries involves recruitment of smooth muscle cells to produce all or none Ca 2 signals

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1 Research Graded 1 -adrenoceptor activation of arteries involves recruitment of smooth muscle cells to produce all or none Ca 2 signals W.-J. Zang, 1,2 C. W. Balke, 1,3 W. G. Wier 1 1 Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore St. Baltimore, MD 21201, USA 2 On leave from Division of Cardiovascular Physiology and Pharmacology, Medical College of Xian Jiaotong University, Xian, , P.R.C. 3 Division of Cardiology, University of Maryland School of Medicine, 655 West Baltimore St. Baltimore, MD 21201, USA Summary Confocal laser scanning microscopy and Fluo-4 were used to visualize Ca 2 transients within individual smooth muscle cells (SMC) of rat resistance arteries during 1 -adrenoceptor activation. The typical spatio-temporal pattern of [Ca 2 ] in an artery after exposure to a maximally effective concentration of phenylephrine (PE, 10.0 M) was a large, brief, relatively homogeneous Ca 2 transient, followed by Ca 2 waves, which then declined in frequency over the course of 5 min and which were asynchronous in different SMC. Concentration Effect (CE) curves relating the concentration of PE (range: 0.1 M to 10.0 M) to the effects (fraction of cells producing at least one Ca 2 wave, and number of Ca 2 waves during 5 min) had EC 50 values of 0.5 M and 1.0 M respectively. The initial Ca 2 transient and the subsequent Ca 2 waves were abolished in the presence of caffeine (10.0 mm). A repeated exposure to PE, 1.5 min after the first had ended, elicited fewer Ca 2 waves in fewer cells than did the initial exposure. Caffeine-sensitive Ca 2 stores were not depleted at this time, however, as caffeine alone was capable of inducing a large release of Ca min after PE. In summary, the mechanism of a graded response to graded 1 -adrenoceptor activation is the progressive recruitment of individual SMC, which then respond in all or none fashion (viz. asynchronous Ca 2 waves). Ca 2 signaling continues in the arterial wall throughout the time-course (at least 5 min) of activation of 1 -adrenoceptors. The fact that the Ca 2 waves are asynchronous accounts for the previously reported fall in arterial wall [Ca 2 ] (i.e. spatial average [Ca 2 ] over all cells). INTRODUCTION Contraction of vascular smooth muscle in response to 1 -adrenceptor agonists, such as phenylephrine (PE), involves both an increase in [Ca 2 ] and activation of protein phosphorylation cascades that may increase the Ca 2 -sensitivity of force, as well as activation of possible Ca 2 -independent mechanisms of force [1]. Increases of intracellular [Ca 2 ] in response to PE are thought to result from release of Ca 2 from intracellular stores (SR) and Received 26 October 2000 Revised 28 December 2000 Accepted 28 December 2000 Published online 8 March 2001 Correspondence to: W. Gil Wier PhD, Dept. of Physiology, UM,B, Room 525B Howard Hall, 655 West Baltimore St, Baltimore, MD 21201, USA. Tel.: ; gwier001@umaryland.edu increased sarcolemmal Ca 2 influx, a portion of which is dependent on depolarization and a minor portion of which is not [2]. Recently, confocal imaging of Ca 2 in individual smooth muscle cells within the walls of intact arteries has shown that the spatio-temporal patterns of this Ca 2 increase are more complex than previously thought. In particular, it has been shown now that the [Ca 2 ] oscillates, and that changes in Ca 2 propagate as Ca 2 waves during exposure to PE [3 5]. This is opposed to the spatially averaged or whole tissue [Ca 2 ], which rises and then falls smoothly to a maintained lower level [6,7]. Furthermore, in rat venous tissue (inferior vena cava), it has been shown recently that the number of cells producing Ca 2 waves is a graded function of the concentration of phenylephrine [8]. This information has been lacking, however, in arterial tissues, where it had been shown [5] only that the fraction of cells producing Ca 2 327

2 328 W-J Zang, CW Balke, WG Wier oscillations was higher in the presence of phenylephrine than in its absence. The goal of this study, therefore, was to investigate the spatio-temporal pattern of Ca 2 signaling in individual SMC within the walls of intact arteries in more detail than had been done previously, paying particular attention to the recruitment of individual SMC to produce Ca 2 waves. This information will be required to understand exactly how asynchronous Ca 2 waves within individual SMC signal for contraction. Ultimately, contraction is determined by the level of phosphorylation of myosin light chain (MLC), which is both Ca 2 -dependent and Ca 2 -independent [9], but the way in which Ca 2 waves, as opposed to a steady elevation of [Ca 2 ] might be related to this level of phosphorylation is as yet unknown. MATERIALS AND METHODS All experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Male Sprague-Dawley rats weighing g were anesthetized by intra-muscular injection of Nembutal sodium ( mg/kg) and sacrificed by cervical dislocation. The mesenteric arcade was dissected and placed in a cold dissection chamber (50 C) containing a solution (the dissection solution) with the following composition (mm): MOPS, 2; NaCl, 145; KCl, 4.7; CaCl 2, 2; MgSO 4, 1.2; NaH 2 PO 4, 1.2; EDTA, 0.02; pyruvate, 2; glucose, 5; 1.0% albumin (ph 7.4). Third (3rd) and 4th order distal arteries were dissected by methods similar to those described previously [5,10]. They were loaded with Fluo-4 at room temperature for 3 4 h, in the dissection solution (but without albumin) to which had been added 10 M Fluo-4 AM, 1.5% DMSO (v/v), and 0.03% cremophor EL (v/v). Cremophor aids the loading of the arteries with Fluo-4 [2,5]. Arteries were cut in 2 4 mm length, mounted over glass cannulae m diameter and studied under isometric conditions (Fig. 1A). Arteries were maintained in a modified Kreb s solution of the following composition: (mm): NaCl, 112; NaHCO 3, 25.7; KCl, 4.9; CaCl 2, 2.5; MgSO 4, 1.2; KHPO 4, 1.2; glucose, 11.5; HEPES 10, (ph 7.4 at 22 C); and equilibrated with gas of 5% O 2, 5% CO 2. PE (Sigma), ACh (Sigma) and Caffeine (Sigma) were prepared in different concentrations just prior to the experiments. Arteries were studied at room temperature ( 22 C). The confocal microscope consisted of a Bio-Rad MRC 600 imaging system connected to a Nikon Diaphot microscope equipped with a Nikon CFN plan Apochromat x 60 water immersion objective (NA 1.2). Images were stored in a computer at 1 frames s 1 through the use of a video-capture board (PCI 1407, National Instruments Corporation, Austin, TX, USA). An optical section of the arterial wall ( 106 mε70 m, and containing about cells), was obtained every second. A schematic illustration of the method of obtaining these images is in Figures 1A and 1B. From these images, virtual line-scan images (e.g. Fig. 2B) could be obtained, with a temporal resolution of 1 s and duration of up to 8 min. The spatial average [Ca 2 ] could also be obtained, by integration of the image. Images were analyzed using Interactive Data Language (Research Systems, Inc., Boulder, CO, USA). A Digital Video clip (available as Supplementary Material on IDEAL) was constructed from the original data using digital video editing software (Adobe Systems Inc., San Jose, CA, USA). The DV clip may be viewed with the Microsoft Windows Media Player (Microsoft Corp. Redmond, WA, USA). RESULTS Responses of the SMC to high levels of receptor stimulation Under control conditions (i.e. absence of PE), a small fraction of the SMC within the arterial wall exhibited Ca 2 waves (12.7%, 976 cells, 36 arteries). These occurred at a low frequency (0.086w.s ), similar to that reported previously for this tissue [5]. The typical Fig. 1 Illustration of method of recording Ca 2 dependent Fluo-4 fluorescence in intact rat mesentery small arteries. (A) Schematic diagram of an artery (shaded cylinder) mounted over a glass cannula. Cannulae were typically m in outer diameter. The double-headed arrow labeled x indicates the direction and plane of fast bi-directional laser scanning. The single-headed arrow indicates the direction of slow scanning. In actual experiments, the bottom of the artery was scanned, not the top, which has been show here, for ease of illustration. (B) Optical section of the arterial wall, illustrating approximately 25 single vascular smooth muscle cells. Image is of Fluo-4 fluorescence, during a synchronous Ca 2 transient (the cells are more readily visible in this condition than when intracellular [Ca 2 ] is low). The arrows labeled x and y, and the distance scale bar correspond to those similarly labeled in A. Thus, the optical section cuts longitudinally through individual SMC that wrap circumferentially around the artery. The z axis resolution of the confocal microscope was 0.7 m. Images were typically 768ε512 pixels. Images of SMC were obtained midway into the arterial wall, at an approximate depth of 10 m.

3 Graded 1 -adrenoceptor activation 329 changes in Ca 2 in response to application of PE (5.0 M) are illustrated in Figure 2 and the accompanying DV clip (Fig. 2A, Zang et al.avi). At this concentration, PE always elicited an initial synchronous increase of Ca 2 in all the SMC that were visible within the optical section of the arterial wall (Fig. 2Ab). The duration of this increase was somewhat variable from cell to cell, but typically lasted s. Following this, the individual SMC then developed propagating Ca 2 waves that were asynchronous between the individual SMC (Fig 2A, c e). These Ca 2 waves decreased in frequency during the exposure to PE and nearly ceased upon its removal (Fig. 2A, f). These results can be seen more easily in the accompanying video clip (Fig. 2A, Zang et al.avi) in which the entire dataset, consisting of 480 images, taken at 1 s 1, can be viewed (the video clip also reveals a few spontaneous Ca 2 sparks that occurred under control conditions, prior to administration of PE). To examine the Ca 2 waves more easily, we constructed virtual line scan images, of the type shown in Figure 2B. These images were obtained by extracting a single vertical line of pixels from each of the 480 full-frame images of the series shown in Figure 2A, and stacking these lines horizontally, to produce the image shown in Figure 2B, in which time runs from left to right. Thus, the image in Figure 2B illustrates the responses of approximately 30 cells, with 1-s resolution during the exposure to PE. The fact that Ca 2 waves are asynchronous between neighboring SMC is very evident in this image, as well as the fact that different SMC produce the Ca 2 waves at different frequencies. No correlation between the activities of neighboring cells was ever apparent, except during the initial response. These phenomena are also revealed in the line-plots of fluorescence within five selected individual SMC (Fig. 2C). Fig. 2 Spatio-temporal changes in [Ca 2 ] elicited by a high concentration of the 1 -adrenoceptor agonist, phenylephrine, in the individual smooth muscle cells of a rat small artery. (A) Images of Fluo-4 fluorescence before (a), during (b e) and after (f) exposure to phenylephrine (5.0 M). The full sequence of 480 images is also available as a video clip, Fig. 2A Zang et al.avi. Image b shows the initial, brief, homogeneous rise in Ca 2 occurring within seconds of exposure to PE. Images c f show the asynchronous Ca 2 waves within individual SMC. (B) Virtual line-scan image constructed from the same sequence of 480 images. Upward arrowheads in this figure indicate the time of application of PE. Dotted line in Aa gives the position of the virtual scan-line. Ca 2 waves within individual SMC are evident as the periodic increases in fluorescence within each cell. Note that the Ca 2 waves within neighboring cells do not appear to be correlated (i.e. are asynchronous), and that the frequency of the Ca 2 waves declines with time during the maintained presence of phenylephrine. (C) Line-plots of Fluo-4 fluorescence of individual cells obtained from the virtual line-scan image at the places indicated by the arrows. (D) The single trace represents the time course of the spatial average [Ca 2 ] during the sequence of 480 images. The trace is the average Fluo-4 fluorescence within the entire image. This is representative of the recording of Fluo-4 fluorescence that would be obtained without any spatial resolution, i.e. as if Fluo-4 fluorescence had been recorded without imaging.

4 330 W-J Zang, CW Balke, WG Wier The spatial average Ca 2 (fluorescence), which would be equivalent to the signal obtained without spatial resolution (i.e. without imaging) can be created from this data by summing the fluorescence in each image, and the line plot of the time course of this total fluorescence is shown in Figure 2D. The time course of this spatial average Ca 2 is distinctly different from the time course of Ca 2 in any of the individual SMC. After an initial increase, the spatial average Ca 2 declines rapidly to a relatively low level, and then declines continuously throughout the exposure to PE. The decline to a relatively low level reflects the fact that the Ca 2 waves in the individual SMC rapidly become asynchronous and then decline in frequency. The time course of the spatial average Ca 2 we record is remarkably similar to that recorded earlier by others [7]. It is clear, however, that the spatial average Ca 2 is not representative of that in individual SMC. In particular, the individual SMC exhibit a pattern resembling baseline spiking [11]. In this pattern, the [Ca 2 ] between spikes is not elevated significantly. Thus, the Ca 2 signaling in individual SMC is actually quite different from the spatial average Ca 2, which does not represent accurately events within any individual SMC. We note that similar phenomena also exist in populations of non-excitable cells (18). These results illustrate the importance of spatially resolved measurements of intracellular Ca 2. Responses of the SMC at different levels of receptor stimulation It was of interest to determine how the response of the individual SMC might be different at lower levels of receptor stimulation. Responses to a 10-fold lower concentration (0.5 M PE) of agonist are shown in a virtual line-scan image in Figure 3A. The responses of three selected SMC are shown in Figure 3B. At this [PE], not all SMC responded to each exposure to PE in a detectable way. Those that did (e.g. cells marked a and c in Fig. 3B) produced only one or a few Ca 2 waves. Since not all cells responded, it was of interest to determine whether a subsequent exposure to PE at 0.5 M would elicit responses in the same cells or in different ones. The second exposure elicited a smaller response involving fewer cells, with fewer Ca 2 waves. Some of the cells responding were the same ( a in Fig. 3B), some were new ( b in Fig. 3B), and some that responded to the first exposure, failed to respond to the second ( c in Fig. 3B). It appears, therefore, that at these levels of receptor stimulation, the response of the individual SMC is probabilistic. It was important to show that all the cells were, in fact, capable of responding to agonist. Thus, this artery was then exposed to PE at a maximally effective concentration, 10.0 M. All the cells then responded to PE at 10.0 M, although the response Fig. 3 Spatio-temporal changes in [Ca 2 ] elicited by repeated applications of a relatively low concentration of the 1 -adrenoceptor agonist, phenylephrine, in the individual smooth muscle cells of a rat small artery. (A) Virtual line-scan image constructed from a sequence of 480 images obtained during two brief exposures (90 s) to PE at concentrations of 0.5 M and then to PE at 10.0 M. (B) Line-plots of the intensity of Fluo-4 fluorescence. PE applied for 90 s each time at the places indicated by the arrows. was much weaker than would normally be obtained if the artery had been exposed first to PE at 10.0 M (n 152 cells from 4 vessels). This may reflect receptor desensitization. A very clear result of this type of experiment however, is that, in individual SMC, the amplitude of the Ca 2 waves is not dependent on agonist concentration (although smaller Ca 2 waves are sometimes seen, at all agonist concentrations). For example, the cell marked b in Fig. 3B failed to produce any Ca 2 wave to the first application of PE at 0.5 M, but then produced Ca 2 waves of roughly the same amplitude to successive applications of PE at 0.5 M and at 10.0 M. Thus, the responses to 1 -agonists within individual SMC appear to be all or none. The all or none nature of the responses may be related to regenerative processes, such as Ca 2 - induced Ca 2 release, which are involved in the production of propagating Ca 2 waves [14].

5 Graded 1 -adrenoceptor activation 331 Fig. 4 Concentration effect (C E) curves for phenylephrine. (A) Frequency histograms showing the probability that a cell will produce a given number of Ca 2 waves, at different concentrations of PE (0.1 M to 5.0 M, as indicated in the inset of each graph). Data are from 36 arteries. Each artery was exposed to PE only once. (B, C) C E curves summarizing the data shown in (A). The curves relate the concentration of PE (range: 0.1 M to 10.0 M) to the effects (fraction of cells producing at least one Ca 2 wave) (B), and number of Ca 2 waves in such cells during 5 min (C). Data points represent mean values and SEM; at least 100 cells of 4 to 6 arteries were used for each data point. Concentration Effect (C E) curves for phenylephrine These results suggest a process in which the probability that an SMC will produce an all or none response to stimulation by 1 -agonists is random, with the probability depending on agonist concentration. It appeared also that receptor desensitization could be important. We investigated these issues further by obtaining concentration effect (C E) curves over the range of agonist concentration 0.1 M to 10.0 M. Each artery studied was exposed to agonist only once, to avoid possible desensitization. Virtual line-scan images such as those in Figure 2B and Figure 3A were analyzed to obtain the percentage of cells responding to PE at given concentration, with at least one Ca 2 wave. The number of Ca 2 waves was counted in each cell during a 5-min exposure to PE. The results are plotted in Figure 4A as frequency histograms, which give the probability that an individual SMC will produce a given number of Ca 2 waves during the exposure to PE. The frequency histograms show clearly that as the concentration of PE increases, the fraction of cells producing at least one Ca 2 wave increases, as does the number of Ca 2 waves in each cell. The results are presented as standard C E curves in Figure 4B and C. In Figure 4B is plotted the percentage of SMC that produced at least one Ca 2 wave, as a function of [PE]. In Figure 4C is plotted the total number of Ca 2 waves in each SMC as a function of [PE]. The concentration at which 50% of the maximum effect is reached (EC 50 ) is similar for both measures, suggesting that the two quantities are related, as might be expected. Mechanism of the response to phenylephrine As mentioned previously, the frequency of Ca 2 waves tended to decrease during maintained exposure to PE. Subsequent exposure to PE, after initial exposure to a high concentration (10.0 M), resulted in a reduced response (Fig. 5A). These two phenomena could possibly be related. Both could possibly be the result of desensitization, or depletion of Ca 2 stores. To investigate the state of intracellular Ca 2 stores during the action of PE, we used caffeine, which opens ryanodine receptors (RyR),

6 332 W-J Zang, CW Balke, WG Wier Fig. 5 (A) Effects of repeated applications of phenylephrine. PE at 10.0 M was applied twice, as indicated. The responses within individual SMC were significantly attenuated in the second application (results are typical of 142 cells from five arteries). (B) The decrease in the frequency of Ca 2 waves during the continued presence of PE is not due to depletion of a Ca 2 store that is releasable by caffeine. In the continued presence of PE, when Ca 2 waves have declined in frequency, caffeine still triggers a large release of Ca 2 (n 153 cells from 4 vessels). (C) The response to phenylephrine is abolished in the presence of caffeine. (a) Virtual line-scan image. Caffeine (10.0 mm) elicited a large increase in [Ca 2 ]. PE was applied at 10.0 M in the continued presence of caffeine. (b) Line-plot of average Fluo-4 fluorescence (i.e. from all cells in the virtual line-scan image). PE elicited only a small increase in [Ca 2 ] and no Ca 2 waves were evident (n 136 cells from 4 vessels). to cause release of Ca 2 from the sarcoplasmic reticulum (SR). Administration of caffeine alone was able to elicit large increases in intracellular Ca 2, and the effect was repeatable, at intervals of 1.5 min (n 136 cells from four vessels). In the presence of PE, caffeine also elicited a massive release of Ca 2, at a time when Ca 2 waves had died away completely, or when further responses to PE would have been much diminished (Fig. 5B). This result indicates that caffeine-sensitive Ca 2 stores are not depleted of Ca 2 in the presence of PE. A similar result was obtained previously in single isolated rat mesentery artery myocytes [13]. The result there was taken to indicate that the 1 -agonist-releasable Ca 2 store and the caffeinereleasable Ca 2 store are different. Exposure of arteries to caffeine in the absence of PE confirmed that the initial release of Ca 2, and production of any subsequent Ca 2 waves, is dependent on the function of the sarcoplasmic reticulum (SR) (Fig. 5Ca). PE did elicit a small slow increase in Ca 2, perhaps reflecting an effect on Ca 2 entry through receptor operated Ca 2 channels or voltage-gated Ca 2 channels. This effect is seen most easily in the spatial average fluorescence

7 Graded 1 -adrenoceptor activation 333 (i.e. Ca 2 ), illustrated in Figure 5Cb. Another phenomenon evident from that recording was that the level of fluorescence following removal of caffeine undershot the control level. Undershoots in Ca 2 upon removal of caffeine had also been observed previously in single isolated rat mesentery artery myocytes [13] and attributed to enhanced uptake of Ca 2 into an SR that is depleted of Ca 2. Finally, while we expect PE to act specifically on the vascular smooth muscle cells, it is possible that the responses to PE were modified in some way by the endothe lium. Since the arteries we studied were not deliberately de-endothelialized, we determined whether the endothelium was normally functional in our preparation. After our normal procedures for loading arteries with Fluo-4, endothelial cells could be readily visualized by adjusting the plane of focus upward in the specimen [5]. As judged by their shape, and ability to retain Fluo-4, the endothelial cells appeared to be intact. Treatment of arteries preactivated by PE (10.0 M) with acetylcholine (ACh, 10.0 M) reduced the frequency of the Ca 2 oscillations (n 138 cells from four vessels). Thus, we conclude that the arteries we studied had an intact, functional endothelium. Nevertheless, the responses to PE that we observed are similar to those of venous SMC in de-endothelialized inferior vena cava [8]. DISCUSSION Different types of isolated smooth muscle cells exhibit a variety of responses (summarized recently by Savineau & Marthan [12]) to agonists that interact with G proteincoupled surface membrane receptors, including: (1) those types of cells in which the frequency, but not amplitude of Ca 2 oscillations increases with increasing concentration of agonist, (2) those types in which both amplitude and frequency increase with increasing concentration of agonist, and (3) those in which the fraction of cells producing Ca 2 oscillations increases with agonist concentration. The SMC of intact rat mesenteric small arteries appear to fit into the last category, although the previous results [13] on isolated rat mesenteric small artery cells are somewhat different, in that high concentrations of noradrenaline (10 M) produced only a single transient increase in [Ca 2 ] and no subsequent Ca 2 oscillations. In the intact artery, however, the response of individual cells can be said to be probabilistic because it appears to be chance that determines which cells will respond with at least one Ca 2 oscillation to intermediate concentrations of agonist. For example, at PE of 0.5 M, approximately 50% of the cells respond with one or more Ca 2 oscillations (Figs 3&4), but it is a different 50% that respond to a second exposure to PE (Fig. 3). The responses may be said to be all or none in the sense that the Ca 2 oscillations are actually Ca 2 waves, which almost certainly propagate by the regenerative mechanism of Ca 2 induced Ca 2 release and inositol-1,4,5-trisphosphate ([InsP 3 ]) induced Ca 2 release [14]. It should be noted, however, that the responses to PE are not true Ca 2 waves in the strictest sense of the word, since the elevation of Ca 2 can last 10 s or more, far longer than that required for the change in [Ca 2 ] to propagate to the ends of the cell. Our results are similar to those obtained recently in intact veins [8], in which increasing numbers of individual SMC respond to increasing concentrations of phenylephrine (viz. are recruited ) with an increasing number of Ca 2 waves. Thus, evidence is accumulating that in both intact veins and arteries, the concentration effect curves for adrenergic agonists are explained in terms of recruitment of SMC, each of which respond with all or none type responses. Furthermore, previous work [4] established that the response to stimulation of the perivascular nerves of rat-tail artery (which should release the physiological -receptor agonist nor-epinephrine) also elicited asynchronous Ca 2 waves in individual SMC. Neither the tension in isometric arteries and veins nor the total tissue Ca 2 reflects the Ca 2 oscillations within the individual smooth muscle cells (as shown for Ca 2 in Fig. 2). The reason that total tissue Ca 2 does not reflect the Ca 2 oscillations is clear: asynchronous Ca 2 oscillations often sum out-of-phase, and are thus not evident in a recording of Ca 2 that is not spatially resolved. The same explanation may be true for tension development, coupled with the fact that tension develops with a considerable delay after a rise in [Ca 2 ]. To obtain the concentration effect curves for PE, we exposed arteries only once to PE because of receptor desensitization or reductions in the levels of the coupling proteins, G,q and G,i [15,16]. It has been suggested that there are at least two post-receptor desensitizing mechanisms activated by 1 -adrenoceptor agonists: reduction in Ca 2 mobilization and reductions in the sensitivity of the contractile proteins to Ca 2 [17]. Our results indicate strongly that a reduction in intracellular Ca 2 is a major component in the desensitization manifested as reduced responsiveness to receptor stimulation. In this regard, a very interesting result is illustrated in Figure 5B. Although the frequency of Ca 2 waves elicited by a high concentration of PE had declined to very low levels (i.e. only a few cells still produced Ca 2 waves), caffeine was still able to release a very large amount of Ca 2. This suggests the reason for the decline in the frequency of the Ca 2 waves was not depletion of Ca 2 stores. It seems possible that this result could be explained by receptor or G protein desensitization, although a similar result in isolated rat mesentery artery SMC was interpreted to indicate that the nor-adrenaline and caffeine-sensitive Ca 2 stores are not the same [13]. The present results do not allow us to distinguish these two possibilities.

8 334 W-J Zang, CW Balke, WG Wier ACKNOWLEDGMENTS Supported by NIH research grants HL to W. Gil Wier and by Natural Scientific Foundation of China (No and No ) to Wei-Jin Zang. We thank Joseph R.H. Mauban and Christine Lamont for help with the preparation of isolated arteries. REFERENCES 01. Martinez MC, Randriamboavonjy V, Ohlmann P et al. Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca 2 handling of human small arteries. Am J Physiol 2000; 279: H1228 H Nilsson H, Jensen PE, Mulvany MJ. Minor role for Direct Adrenoceptor-Mediated Calcium Entry in Rat Mesenteric Small Arteries. J Vasc Res 1994; 31: Iino M, Kasai H, Yamazawa T. Visualization of neural control of intracellular Ca 2 concentration in single vascular smooth muscle cells in situ. EMBO J 1994; 13 (21): Kasai Y, Yamazawa T, Sakurai T, Taketani Y, Iino M. Endothelium-dependent frequency modulation of Ca 2 signalling in individual vascular smooth muscle cells of the rat. J Physiol 1997; 504.2: Miriel V, Mauban J, Blaustein MP, Wier WG. Local and cellular Ca 2 transients in smooth muscle of pressurized rat resistance arteries during myogenic and agonist stimulation. J Physiol 1999; 518.3: Meininger GA, Zawieja DC, Falcone JC, Hill MA, Davey JP. Calcium measurement in isolated arterioles during myogenic and agonist stimulation. Am J Physiol 1991; 261(3 Pt. 2): H950 H Jiang MJ, Morgan KG. Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle. Pflugers Arch 1989; 413: Ruehlmann DO, Lee C-H, Poburko D, vanbreemen C. Asynchronous Ca 2 waves in intact venous smooth muscle. Circ Res 2000; 86: e72 e Weber LP, Van Lierop JE, Walsh MP. Ca 2 -independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments. J Physiol 1999; 516.3: Duling BR, Gore RW, Dacey Jr RG, Damon DN. Methods for isolation, cannulation, and in vitro study of single microvessels. Am J Physiol 1981; 941: H108 H Thomas AP, Bird GStJ, Hajnoczky G, Robb-Gaspers LD, Putney JW. Spatial and temporal aspects of cellular calcium signaling. FASEB J 1996; 10: Savineau J-P, Marthan R. Cytosolic calcium oscillations in smooth muscle cells. NIPS 2000; 15: Baro I, Eisner DA. Factors controlling changes in intracellular Ca 2 concentration produced by noradrenaline in rat mesenteric artery smooth muscle cells. J Physiol 1995; 482.2: Blatter LA, Wier WG. Agonist-induced [Ca 2 ] i waves and Ca 2 -induced Ca 2 release in mammalian vascular smooth muscle cells. Am J Physiol 1992; 263: H576 H Seasholtz TM, Gurdal H, Wang HY, Cai G, Johnson MD, Friedman E. Heterologous desensitization of the rat tail artery contraction and inositol phosphate accumulation after in vitro exposure to phenylephrine is mediated by decreased levels of Galphaq and Galphai. J Pharmacol Exp Ther 1997; 283(2): Ratz PH. Receptor activation induces short-term modulation of arterial contractions: memory in vascular smooth muscle. Am J Physiol 1995; 269: C417 C Ratz PH. Dependence of Ca 2 sensitivity of arterial contractions on history of receptor activation. Am J Physiol 1999; 277: H1661 H Thomas AP, St. J. Bird G, Hajnoczky G, Robb-Gaspers LD, Putney JW. Spatial and temporal aspects of cellular calcium signaling. FASEB J 1996; 10: Supplementary Material is available on IDEAL