AJH 1997;10:

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1 AJH 1997;10: Augmented Contributions of Voltage-Gated Ca 2 Channels to Contractile Responses in Spontaneously Hypertensive Rat Mesenteric Arteries Kyoko Matsuda, Irina Lozinskaya, and Robert H. Cox The observation that organic Ca 2 channel blockers are more effective in lowering blood pressure and peripheral resistance in hypertensive compared to normotensive subjects suggests that there is a greater contribution from voltage-gated Ca 2 channels (Ca L ) to vascular force maintenance in hypertensive arteries. This study tests this hypothesis by comparing the effects of Bay k 8644 and nisoldipine on basal force development, contractile responses to norepinephrine and serotonin, and Ca 2 currents (I Ca ) in mesenteric artery (MA) from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). MA rings were used to record isometric contractions at L max. Single cells were isolated by collagenase plus elastase for measurement of Ca L properties by patch-clamp methods. Contractile responses to Bay k 8644 were larger and more sensitive in SHR than WKY, and were larger in endothelium-denuded compared to intact rings. In SHR, the addition of 10 nmol/l Bay k 8644 increased contractile sensitivity to norepinephrine (NE) and serotonin (5HT), and increased maximum response to 5HT. In WKY, 10 nmol/l Bay k 8644 produced a small increase in 5HT sensitivity with no effect on maximum response, and had no effect on NE responses. In the presence of 1 mol/l nisoldipine, the maximum response and the sensitivity to both NE and 5HT were decreased in both WKY and SHR with the inhibitory effects of nisoldipine being larger in SHR than WKY. Peak I Ca was larger in SHR, and current-voltage curves were shifted toward more negative voltages compared to WKY. Bay k 8644 increased I Ca in both WKY and SHR myocytes with no apparent difference in the magnitude of its effect when expressed as a percent of control I Ca. These results suggest that Ca L contribute significantly to tonic force maintenance as well as to agonist responses in MA from both WKY and SHR, but with a much larger contribution in SHR. Differences in the sensitivity of Ca L to Bay k 8644 were not responsible for the differences in contractile responses to this agonist. Am J Hypertens 1997;10: American Journal of Hypertension, Ltd. KEY WORDS: Hypertension, arterial smooth muscle contractions, Ca 2 currents, norepinephrine, serotonin, endothelium, nisoldipine, Bay k Received January 22, Accepted April 22, From the Bockus Research Institute, The Graduate Hospital, and Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania. This study was supported by Research Grant HL from the U.S. Public Health Service. Address correspondence and reprint requests to, Robert H. Cox, PhD, Bockus Research Institute, The Graduate Hospital, One Graduate Plaza, Philadelphia, PA 19146; rcox@mail.med.upenn.edu 1997 by the American Journal of Hypertension, Ltd /97/$17.00 Published by Elsevier Science, Inc. PII S (97)

2 1232 MATSUDA ET AL AJH NOVEMBER 1997 VOL. 10, NO. 11, PART 1 An increase in intracellular Ca 2 is generally regarded to be the primary mechanism by which activation of smooth muscle is coupled to contraction. 1 The increase in cytoplasmic free Ca 2 that activates vascular smooth muscle occurs as a result of both intracellular Ca 2 release and extracellular Ca 2 influx. 1,2 Agonist stimulation of smooth muscle results in activation of phospholipase C, which increases the production of the second messenger inositol 1,4,5-trisphosphate (IP 3 ). 3 IP 3 promotes the release of Ca 2 stores in the sarcoplasmic reticulum with the subsequent increase in intracellular free Ca 2, phosphorylation of myosin light chains, and activation of contraction. 1,4,5 However, the production of IP 3, the Ca 2 transient, and the myosin light chain phosphorylation associated with agonist activation are all transient. 3,4,6,7 In the absence of extracellular Ca 2 influx the steady, tonic component of smooth muscle contraction (ie, force maintenance) cannot be sustained. 8 Also, the tonic level of contraction has been shown not to depend on the degree to which the intracellular Ca 2 stores are filled before activation. 9 Thus, sustained Ca 2 influx is required for sustained contractions in smooth muscle. 10 Van Breemen and co-workers 2 demonstrated three separate pathways for Ca 2 influx in vascular smooth muscle: leak channels, receptor-operated channels (ROC), and voltage-operated channels (VOC). This classification was based on the observation that ROC and VOC could be inhibited and activated separately, whereas the leak Ca 2 influx was present in the absence of activation. 11 This classification was consistent with an earlier classification of excitation contraction coupling processes as electromechanical (membrane potential dependent) and pharmacomechanical (membrane-potential independent) by Somlyo and Somlyo. 12 Utilization of the two activatable pathways (ROC and VOC) during activation by physiologic means are not separate but overlap exists. For example, receptor-mediated stimulation of smooth muscle results in membrane depolarization 13 as a result of activation of Ca 2 -dependent Cl channels 14 and inhibition of voltage-dependent K channels. 15 This membrane depolarization activates voltage-gated Ca 2 channels, which contributes to the sustained contractile response. 16 Organic Ca 2 channel blockers have been shown to inhibit basal tone 17 and the Ca 2 channel agonist Bay k 8644 has been shown to produce larger tonic contractions in hypertensive arterial smooth muscle in vitro. 18,19 Recently, we reported that currents carried by L-type Ca 2 channels (Ca L ) are larger and their voltage-dependence shifted in the negative direction in mesenteric arteries (MA) from spontaneously hypertensive rats (SHR) compared to Wistar-Kyoto rats (WKY). 20 Furthermore, Ca 2 channel blockers are known to be effective antihypertensive agents. 21 These results suggest that under normal conditions Ca L may be active in hypertensive smooth muscle, and may provide a significant contribution to force maintenance and peripheral resistance in hypertensive smooth muscle. The extent to which Ca L are involved differentially in agonist-mediated contractions in WKY and SHR, however, is not clear. It was the purpose of this study to test the hypothesis that Ca L provides a greater contribution to agonist-mediated contraction in hypertensive compared to normal arterial smooth muscle. METHODS The mesenteric artery and its branches were obtained from 12-week-old male WKY and SHR rats. The main MA was cut into 3-mm long ring segments for use in contractile studies. Small MAs were used for the isolation of single cells by collagenase and elastase dissociation. 20 Ca 2 currents (I Ca ) were measured in these cells using whole cell patch clamp methods. Contractile Studies For these studies, the rings were mounted in vitro at their optimum length for force development in a temperature-controlled bath containing a physiologic salt solution (PSS) at 37 C and continuously aerated with 95% O 2 /5% CO Some preparations were gently rubbed to remove the endothelium before mounting. 22 After a 2- to 3-h equilibration, duplicate responses to a 120 mmol/l K PSS solution were determined, where K was substituted for Na on an equimolar basis. This response was used as a test of tissue viability and for normalization of agonist contractile responses. In one set of experiments, responses to the cumulative addition of the L-type Ca 2 channel agonist Bay k 8644 were obtained in segments either with or without endothelium. In a separate set of experiments, cumulative concentration response studies were performed using norepinephrine (NE) or serotonin (5HT) in different tissues. After the initial control responses to NE or 5HT, the baths were drained and rinsed twice. Either Bay k 8644 (10 nmol/l) or nisoldipine (1 mol/l) was added to the bath for 30 min, then the dose response test was repeated. The tests were always performed using three rings per animal. One ring was not exposed to either dihydropyridine and served as a time control. Recovery periods of at least 1 h between drug tests were used. Force was divided by wall cross-sectional area and expressed as active stress as previously described. 22 Peak values of stress were determined at each drug concentration and also expressed as percentage of the maximal response. Values of active stress and percent response were averaged at each drug concentration for the two groups (WKY and SHR).

3 AJH NOVEMBER 1997 VOL. 10, NO. 11, PART 1 Ca 2 CHANNELS AND CONTRACTION IN SHR 1233 Cell Isolation Myocytes were isolated enzymatically from small MAs using methods previously described by us. 20 Individual branches were removed, cut open longitudinally, and incubated in a Ca 2 -free solution at 37 C for about 40 min. The branches were then cut into small pieces and collagenase (250 U/mL) plus elastase (25 U/mL) were added to the incubation solution for about 30 min at 37 C. The tissue and enzyme solution were separated by filtering through 210- m mesh, washed with 1 ml of enzyme-free buffer, and triturated gently using a Pasteur pipette. Released cells were separated from the tissue by filtering through the 210- m mesh. Electrophysiological Methods An aliquot of cells was transferred to a chamber placed on the stage of an inverted microscope (Nikon Diaphot, Melville, NJ) and allowed to adhere to the chamber s glass bottom. Perfusion with a Ca 2 -free solution was initiated to remove debris and loosely attached cells from the chamber. The [Ca 2 ] of the perfusate was slowly increased to 2 mmol/l to avoid cell damage. Membrane currents were recorded using the whole cell, patchclamp configuration 23 at room temperature (about 22 to 24 C). Micropipettes (2 to 3 M resistance) were made from capillary tubing (WPI Kwik-fil, Sarasota, FL) using a programmable puller (P-80/PC; Sutter Instruments, San Rafael, CA) and fire polished. Series resistance and capacitance compensation were adjusted maximally using a patch-clamp amplifier with a 100 M head stage (model 8900; Dagan, Minneapolis, MN). Experimental protocols were controlled using a computer (466/L; Dell, Austin, TX) and PCLAMP software (version 5.5.1; Axon Instruments, Foster City, CA). Current signals were converted from analog to digital form at a sampling rate of 10 khz using a Labmaster A/D board (Axon Instruments) and stored in the computer for analysis. Multiple responses (n 5) to hyperpolarizing voltage-clamp steps (20 mv) were obtained for each protocol, averaged, and used to provide capacitance and leak compensation of the raw data. Experimental current records were analyzed using PCLAMP software. Procedures Cell break-in was accomplished by gentle suction at a holding potential of 60 mv. Membrane potential was stepped at 10-sec intervals from 60 to 0 mv for 3 to 5 min during cell dialysis with the pipette solution until I Ca stabilized. Current-voltage measurements were then performed from a holding potentials of 90 mv. Voltage-clamp steps 75 msec long were applied from 60 to 40 mv in 10-mV increments every 10 sec. After control measurements, cells were exposed to 10 nmol/l then to 1 mol/l Bay k 8644 added to the perfusate sequentially, and the same measurements were repeated at each concentration. The response to the agonist was monitored using a voltage-clamp step from 60 to 0 mv every 15 sec until a steady response was achieved. Chemicals and Solutions The PSS used for contractile studies had the following composition (in mmol/ L): 114 NaCl, 4.5 KCl, 2.5 CaCl 2, 1.2 MgSO 4, 24 NaHCO 3, 1.2 NaH 2 PO 4, 2.4 Na 2 HPO 4, and 11 dextrose with ph of 7.4 (KOH) and osmolality of mosm/l. All drugs were freshly made in double distilled water and added to the baths with a volume dilution that never exceeded 1000:1. The incubation buffer for enzymatic cell isolation and the external solution for patch-clamp studies had the following composition (in mmol/l): 140 NaCl, 5 KCl, 1 MgCl 2, 10 HEPES, and 10 glucose, with ph 7.4 (with NaOH) and had an osmolality of mosm/l. The solution used to fill the patch pipettes had the following composition (in mmol/l): 100 CsCl, 20 tetraethylammonium chloride (TEACl), 5 NaCl, 5 MgATP, 10 HEPES, and 10 1,2,-bis(2-aminophenoxy)ethane- N,N,N,N -tetraacetic acid (BAPTA) at ph 7.2 (with CsOH), and had an osmolality of mosm/l. Collagenase was purchased from Worthington Biochemical (CLS3, Freehold, NJ). Elastase was purchased from ICN Pharmaceuticals (porcine pancreas, Cleveland, OH). Bay k 8644 was obtained from Research Biochemicals Int. (Natick, MA), and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Nisoldipine was a gift from Miles Laboratories Inc. (West Haven, CT). Statistical Analysis Statistical comparisons of membrane currents were performed using a two-way analysis of variance with repeated measures for unpaired data using the StatWorks (Cricket Software, Philadelphia, PA) application on a Macintosh computer (PowerMac 7100; Apple, Cupertino, CA). Probability values.05 were considered to be significant. Average values for the two groups are given for each procedure as mean 1 SEM. RESULTS The cumulative addition of Bay k 8644 caused a large dose-dependent contractile response in MAs from SHR that was significantly greater than that in WKY as shown in Figure 1. This response was augmented after endothelium removal in terms of threshold, sensitivity, and maximum response. In the presence of the endothelium, the maximum response to Bay k 8644 in SHR was about 55% of the response to 120 mmol/l KCl and occurred at 1 mol/l. When the endothelium was removed, the maximum response was equal to that of 120 mmol/l KCl and occurred at 0.3 mol/l. In contrast, responses to Bay k 8644 in WKY tissues were smaller in both endothelium-intact and endothelium-denuded preparations. As in SHR mesenteric arteries, removal of the endothelium enhanced responses to Bay k 8644 in WKY. Contractions to 120

4 1234 MATSUDA ET AL AJH NOVEMBER 1997 VOL. 10, NO. 11, PART 1 FIGURE 1. Cumulative dose response curves for Bay k E( ) and E( ) represents endotheliumintact and endothelium-denuded preparations, respectively. A and B: Peak contractions were determined at each concentration and expressed as a percentage of maximum contraction to 120 mmol/l KCl. C: Maximum response to Bay k 8644 is expressed as active stress. Mean contractile responses to Ca 2 channel opener were significantly greater in SHR compared to WKY with a threshold of 10 nmol/l and with larger responses in endothelium-denuded preparations. Contractions to 120 mmol/l KCl were not significantly different. (*) Significant differences (P.05). Values are the mean SEM for 9 to 12 preparations. mmol/l KCl were not significantly different with or without endothelium between WKY and SHR. The effects of augmenting Ca L activity on NE dose response relations were determined by pretreating tissues with 10 nmol/l Bay k These responses were performed after removal of the endothelium in the presence of 0.1 mol/l propranolol (to block adrenoceptors) and 0.1 mol/l desipramine (to block neuronal norepinephrine uptake). Tissues were pretreated with Bay k 8644 for 30 min before the initiation of the cumulative NE responses. As shown in Figure 2, Bay k 8644 had no significant effect on NE dose responses relations in the WKY, but produced a significant augmentation of responses in the SHR. As the maximum response to NE was not affected by Bay k 8644, the augmentation in SHR amounted to a leftward shift in the dose response relation. The effects of augmenting Ca L activity on serotonin dose response relations were also determined using endothelium-denuded preparations preexposed to 10 nmol/l Bay k As shown in Figure 3, Bay k 8644 increased the sensitivity to 5HT in both WKY and SHR, but with a larger effect in SHR. The effect of Bay k 8644 in WKY was significant only at low concentrations of 5HT, whereas the effect in SHR appeared as a parallel shift in the dose response relation to the left. FIGURE 2. Effects of Bay k 8644 on norepinephrine (NE) responses. Responses were obtained in the presence of 0.1 mol/l propranolol (to block -adrenoceptors) and 0.1 mol/l desipramine (to block norepinephrine uptake) after removal of endothelium. A and B: Cumulative responses to NE were normalized to the maximum response before averaging. C: Summary of maximum stress responses to NE. Only SHR preparations showed augmented contractile sensitivity to NE in the presence of 10 nmol/l Bay k *Significant differences (P.05). Values are the mean SEM for 5 to 11 preparations.

5 AJH NOVEMBER 1997 VOL. 10, NO. 11, PART 1 Ca 2 CHANNELS AND CONTRACTION IN SHR 1235 FIGURE 3. Effects of Bay k 8644, on serotonin (5HT) responses. The effects of 10 nmol/l Bay k 8644 were determined in endothelium-denuded preparations. A and B: Responses were normalized to the maximum stress before averaging. C: Summary of maximum responses to 5HT. Bay k 8644 increased the sensitivity to 5HT with a larger response in SHR than WKY. Maximum response was also augmented in SHR preparations. *Significant differences (P.05). Values are the mean SEM for 6 to 12 preparations. The maximum response to 5-HT was significantly increased in SHR, but not in WKY, as indicated in Figure 3C. The contribution of the normal level of Ca L activity to NE dose response relations was assessed by pretreating endothelium-denuded preparations with 1 mol/l nisoldipine. NE responses were then obtained in the presence of 0.1 mol/l propranolol and 0.1 mol/l desipramine. As shown in Figure 4, the NE dose response curves were significantly shifted to the right in both groups by nisoldipine but with a much larger effect in the SHR. Nisoldipine also decreased the maximum response to NE in both WKY and SHR, also with a larger effect in SHR. The normal contribution of Ca L to serotonin dose response relations was also determined in endothelium-denuded preparations. In the presence of 1 mol/l nisoldipine, the dose response for 5HT was shifted to the right in both WKY and SHR, with a much larger effect in the SHR as shown in Figure 5. The effect of nisoldipine in SHR was largest at lower 5HT concentrations. In addition, nisoldipine reduced the maximum stress responses to 5HT in both WKY and SHR, also with a larger effect in the SHR (WKY 26 5% v SHR 72 8%). The effects of Bay k 8644 on I Ca were determined using myocytes isolated from mesenteric arteries of the same animals in which the contractile responses FIGURE 4. Effects of nisoldipine on NE responses. Responses to cumulative addition of NE was performed after treatment with 1 mol/l nisoldipine. Responses were obtained in endothelium-denuded preparations in the presence of 0.1 mol/l propranolol and 0.1 mol/l desipramine. A and B: Responses were normalized to the maximum responses before averaging. C: Summary of maximum active stress responses to NE in the presence of 1 mol/l nisoldipine. Sensitivity to NE was decreased in the presence of nisoldipine with a larger effect in SHR compared to WKY. Nisoldipine produced a larger decrease in the maximum responses to NE in preparations from SHR. *Significant differences (P.05). Values are the mean SEM for 5 to 10 preparations.

6 1236 MATSUDA ET AL AJH NOVEMBER 1997 VOL. 10, NO. 11, PART 1 FIGURE 5. Effects of nisoldipine on serotonin (5HT) responses. A and B: Responses were normalized to the maximum response before averaging. C: Summary of maximum active stress responses to 5HT with 1 mol/l nisoldipine. Nisoldipine produced a larger decrease in the maximum response and in sensitivity to 5HT in preparations from SHR. *Significant differences (P.05). Values are the mean SEM for 7 to 9 preparations. were determined. Under control conditions, I Ca current density (I Ca divided by cell capacitance to normalize for differences in cell size) was significantly larger in myocytes from SHR compared to WKY ( v pa/pf) as previously reported. 24 As shown in Figure 6, both 10 nmol/l and 1 mol/l Bay k 8644 increased I Ca in WKY and SHR, and shifted the current-voltage relations to the left (in the hyperpolarizing voltage direction). At each concentration of Bay k 8644, I Ca remained significantly larger in SHR compared to WKY. However, no differences existed in I Ca responses to Bay k 8644 between WKY and SHR at either concentration when represented as percentage changes from initial values. The percentage increases in maximum I Ca were as follows: at 10 nmol/l: WKY 58 13% (n 6) and SHR 48 7% (n 7); at 1 mol/l: WKY % (n 6) and SHR 112 6% (n 7). The hyperpolarizing voltage shift produced by Bay k 8644, shown in Figure 7, was quantitated by determining the voltage on the increasing limb of the current-voltage curve (in the negative voltage range between 60 and 0 mv) where current was 50% of the maximum value. Changes in the average value of this voltage from control for the various groups was as follows: at 10 nmol/l: WKY % (n 6) and SHR % (n 7); at 1 mol/l: WKY % (n 6) and SHR % (n 7). The differences in values of voltage for half maximal current between WKY and SHR were statistically significant for both 10 nmol/l and 1 mol/l Bay k FIGURE 6. Effects of Bay k 8644 on calcium currents. No differences in the percentage increase in I Ca were found for mesenteric artery myocytes from WKY (n 11) and SHR (n 10). Responses were as follows: at 10 nmol/l: WKY 58 13% and SHR 48 7%; at 1 mol/l: WKY % and SHR 112 6%. I Ca was measured in freshly isolated myocytes at voltages from 60 to 40 mv in 10 mv, 100-msec steps from a holding potential of 90 mv with [Ca 2 ] o 2 mmol/l and [BAPTA] i 10 mmol/l at room temperature. After control measurements, cells superfused with Bay k 8644 and voltage steps were repeated. Peak currents were normalized by cell capacitance before averaging. Symbols are means and are defined in the figure with vertical bars 1 SEM.

7 AJH NOVEMBER 1997 VOL. 10, NO. 11, PART 1 Ca 2 CHANNELS AND CONTRACTION IN SHR 1237 FIGURE 7. Voltage dependence of Bay k 8644 effects on I Ca. Values of I Ca were divided by the maximum value for each cell before averaging at each voltage under control conditions (open circles), and after the addition of 10 nmol/l (closed circles) and 1 mol/l (inverted triangles) Bay k 8644 to the perfusate. Symbols are means and vertical bars are 1 SEM. Number of observations are the same as in Figure 6. DISCUSSION The results of this study provide evidence to support the hypothesis that L-type Ca 2 channels provide a greater contribution to agonist-stimulated contractile responses in mesenteric arteries of SHR compared to WKY. In agreement with results of previous investigations, 18,19 we found larger contractile responses in SHR to the L-type Ca 2 channel activator Bay k 8644 in the presence as well as the absence of the endothelium. This finding is consistent with the differences in the properties of Ca L that we have reported in this tissue. 20,24 We found that Ca 2 currents were larger and activated at more negative voltages in SHR compared to WKY. Both of these characteristics could contribute to the larger responses in SHR. In addition, a more depolarized resting membrane potential of arterial smooth muscle reported in SHR could contribute to the larger responses to Ca L activation in this group. 25,26 Together, these results suggest that Ca L are more active at prevailing membrane potential in SHR compared to WKY. Enhancing the activity of L-type Ca 2 channels with Bay k 8644 or inhibiting it with nisoldipine produced substantial effects on contractile responses to NE and 5HT that were quantitatively different between agonists and strains. In SHR, responses to NE were shifted to the left by Bay k 8644 in parallel to the control curve with no effect on maximum response. It has been shown that NE depolarizes the membrane potential in arterial smooth muscle. 13,27 This effect in the presence of augmented Ca L activity could be responsible for the augmented contractile responses to NE in the SHR. It should be noted that the maximum response to NE was not altered. This is probably attributable to the depolarizing effect of NE on membrane potential saturates with increasing concentrations of NE. 27 The lack of effect of Bay k 8644 on NE responses in WKY suggests that Ca L cannot be further activated by the agonist, membrane potential depolarization is smaller or is insufficient to activate Ca L, or the 5HT buffers the augmented Ca 2 influx to a greater extent, 28 preventing a contractile effect. Nisoldipine pretreatment depressed the contractile response to NE in both WKY and SHR with a larger effect in SHR. The effect of nisoldipine at low concentrations of NE in SHR mesenteric arteries was the most dramatic, and could be attributable to either a decrease in the contribution of Ca L to Ca 2 mobilization per se or to a reduction in the Ca 2 content of the 5HT. 28 Because a contribution of Ca L could not be augmented in the WKY by Bay k 8644 it is likely that a reduction in 5HT Ca 2 content could be the primary reason for the decreased responses in WKY. In the SHR, however, because the Ca L effects on NE responses could be augmented and the effects of nisoldipine were larger than in WKY, it is likely that an inhibitory effect on both Ca 2 influx and 5HT release (ie, content) may play a role in the depressed NE responses in SHR. The effects of modulation of Ca L on 5HT responses in WKY and SHR were generally similar to the effects on NE responses. The only differences were that the effects on 5HT responses were smaller than the effects on NE responses, and Bay k 8644 had a small but significant effect on 5HT responses in WKY. In addition, there was a much larger inhibitory effect of nisoldipine on the maximum response to serotonin in SHR compared to WKY than on maximum NE responses in the two groups. This suggests that 5HT may use Ca L influx for Ca 2 mobilization to a larger extent than NE. The above discussion was based on the implicit assumption that the contribution of Ca L to NE and 5HT contractile responses was only the result of

8 1238 MATSUDA ET AL AJH NOVEMBER 1997 VOL. 10, NO. 11, PART 1 changes in open probability secondary to changes in membrane potential. In fact, both NE and 5HT have been shown to augment Ca L currents probably through a G-protein-mediated pathway Differences in the magnitude of agonist augmentation of Ca L current could contribute to the differences in the effects of the dihydropyridine on agonist-induced contractions between the WKY and SHR but this has not been studied to date. In summary, L-type Ca 2 channels provide a larger contribution to contractile function in arterial smooth muscle under normal conditions in SHR compared to WKY. The larger effect of Bay k 8644 on contractile responses in SHR is not the result of a greater effect of the agonist on Ca 2 channels but may be attributable to differences in the voltage-dependence of the Ca 2 currents or the resting membrane potential. However, it remains to be determined whether the result of these experiments can be extrapolated to human hypertension. REFERENCES 1. Somlyo AP, Somlyo AV: Signal transaction and regulation in smooth muscle. Nature 1994;372: van Breemen C, Cauvin C, Johns A, et al: Ca 2 regulation of vascular smooth muscle. Fed Proc 1986;45: Berridge MJ: Inositol trisphosphate, calcium, lithium and cell signaling. J Am Med Assoc 1989;262: Dillon PF, Aksoy MO, Driska SP, Murphy RA: Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 1981;211: Kamm KE, Stull JT: The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol 1985;25: Murphy RA: Muscle cells of hollow organs. News in Physiological Science 1988;3: Morgan KG, Suematsu E: Effects of calcium on vascular smooth muscle tone. Am J Hyperten 1990;3:291S 298S. 8. Daly CL, Dunn WR, McGrath JC, et al: An examination of the sources of calcium for contractions mediated by postjunctional 1 - and 2 -adrenoceptors in several blood vessels isolated from the rabbit. Br J Pharmacol 1990;99: Ratz PH, Murphy RA: Contributions of intracellular and extracellular Ca 2 pools to activation of myosin phosphorylation and stress in swine carotid media. Circ Res 1987;60: Khalil RA, van Breemen C: Sustained contraction of vascular smooth muscle: calcium influx or C-kinase activation. J Pharmacol Exp Ther 1988;244: Cauvin C, Johns A, Yamamoto MK, et al: Ca 2 movements in vascular smooth muscle and their alterations in hypertension, in Kwan CY (ed): Membrane Abnormalities in Hypertension, Vol. 1. Boca Raton, FL, CRC Press, 1989, pp Somlyo AP, Somlyo AV: Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J Pharm Exp Ther 1968;159: Haeusler G: Contraction, membrane potential and calcium fluxes in rabbit pulmonary arterial muscle. Fed Proc 1983;42: Amedee T, Benham CD, Bolton TB, et al: Potassium, chloride and non-selective cation conductances opened by noradrenaline in rabbit ear artery cells. J Physiol 1990;423: Gelband CH, Hume JR: [Ca 2 ] i inhibition of K channels in canine renal artery: novel mechanism for agonist-induced membrane depolarization. Circ Res 1994; 77: Nelson MT, Patlak JB, Worley JF, Standen NB: Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol 1990;28: C3 C Asano M, Masuzawa-Ito K, Matsuda T: Charybdotoxin-sensitive K channels regulate the myogenic tone in the resting state of arteries from spontaneously hypertensive rats. Br J Pharmacol 1993;108: Traub O, Webb RC: Angiotensin-converting enzyme inhibition during development alters calcium regulation in adult hypertensive rats. J Pharmacol Exp Therap 1993;267: Watts SW, Traub O, Webb RC: Effects of ramipril on contractile oscillations in arteries from genetically hypertensive rats. Clin Exp Hypertens 1994;16: Cox RH, Lozinskaya IM: Augmented calcium currents in mesenteric artery branches of the spontaneously hypertensive rat. Hypertension 1995;26: Fleckenstein AG, Fleckenstein-Grun D, Frey M, Zorn J: Calcium antagonism and ACE inhibition. Two outstandingly effective means of interference with cardiovascular calcium overload, high blood pressure, and arteriosclerosis in spontaneously hypertensive rats. Am J Hypertens 1989;2: Cox RH, Haas KS, Moisey DM, Tulenko TN: Effects of endothelium regeneration on canine coronary artery function. Am J Physiol 1989;257:H1681 H Hamill OP, Marty A, Neher E, et al: Improved patchclamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 1981;391: Lozinskay IM, Cox RH: Effects of age on Ca 2 currents in small mesenteric artery myocytes from WKY and SHR. Hypertension 1997;29: Martens JR, Gelband CH: Alterations in rat interlobar artery membrane potential and K channels in genetic and nongenetic hypertension. Circ Res 1996;79: Stekiel WJ: Electrophysiological mechanisms of force development by vascular smooth muscle membrane in hypertension, in Lee R (ed): Blood Vessel Changes in Hypertension, Vol. II. CRC Press, Boca Raton, FL, 1989, pp Trapani A, Matsuki N, Abel PW, Hermsmeyer K: Norepinephrine produces tension through electromechanical coupling in rabbit ear artery. Eur J Pharmacol 1981; 72: van Breemen C, Chen Q, Laher I: Superficial buffer

9 AJH NOVEMBER 1997 VOL. 10, NO. 11, PART 1 Ca 2 CHANNELS AND CONTRACTION IN SHR 1239 barrier function of smooth muscle sarcoplasmic reticulum. Trends Pharmacol Sci 1995;16: Benham CD, Tsien RW: Noradrenaline modulation of calcium channels in single smooth muscle cells from rabbit ear artery. J Physiol 1988;404: Droogmans G, Declerck I, Casteels R: Effects of adrenergic agonists on Ca 2 -channel currents in single smooth muscle cells. Pflügers Arch 1987;409: Worley JF, Quayle JM, Standen NB, Nelson MT: Regulation of single calcium channels in cerebral arteries by voltage, serotonin, and dihydropyridines. Am J Physiol 1991;261:H1951 H Lepretre N, Mironneau J: 2 -Adrenoceptors activate dihydropyridine-sensitive calcium channels via G i -proteins and protein kinase C in rat portal vein myocytes. Pflügers Arch 1994;429: