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Endocrinology Vol. 142, No. 7 2865-2873
Copyright © 2001 by The Endocrine Society

ARTICLES

L-Type Ca2+ Channel Regulation by Pituitary Adenylate Cyclase-Activating Polypeptide in Vascular Myocytes from Spontaneously Hypertensive Rats1

B. Li2, C. L. Chik2, A. K. Ho and E. Karpinski

Departments of Physiology and Medicine (C.L.C.), University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Dr. E. Karpinski, 7–35 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: ed.karpinski{at}ualberta.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pituitary adenylate cyclase-activating polypeptide (PACAP), a vasoactive peptide, modulates the L-type Ca2+ channel current (L channel current) in vascular smooth muscle cells (VSMC) through activation and integration of two intracellular pathways, protein kinase A and protein kinase C (PKC). In the present study we compared the effects of PACAP on the L channel current in VSMC from the spontaneously hypertensive rats (SHR) and normotensive controls, Wistar Kyoto rats (WKY). We found that compared with WKY, VSMC from SHR had a higher L channel current density. Stimulation by PACAP (10 nM) caused an increase in the amplitude of the whole cell current and prolonged open time in VSMC from SHR and WKY, with the increase greater in SHR. These effects of PACAP on the L channel current was mimicked by an activator of PKC. In contrast, PACAP caused a smaller increase in cAMP accumulation in VSMC from SHR than WKY, and there was no difference in the inhibitory effect of 8-bromo-cAMP on the L channel current from both type of cells. The greater increase in amplitude of the L channel current by PACAP in VSMC from SHR persisted in the presence of adenosine cyclic 3',5'-monophosphothioate, Rp-isomer, a cAMP antagonist, but not calphostin C, a PKC inhibitor. Taken together, our results show an increase in L channel current density and an enhanced PACAP effect on the L channel current in VSMC from SHR compared with WKY. This difference in PACAP response appears to be predominately secondary to an increased PKC sensitivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY ADENYLATE cyclase-activating polypeptide (PACAP) is a neuropeptide that belongs to the vasoactive intestinal peptide/secretin/glucagon peptide family (1). PACAP, like vasoactive intestinal peptide, has significant vasoactive effects that depend on the vascular bed studied (2, 3, 4, 5, 6, 7, 8, 9). The importance of PACAP as an vasoactive peptide is supported by observations that PACAP-containing neurons have been found in arteries (6), and PACAP receptors have been localized in membranes prepared from blood vessels (3, 5). In addition, PACAP can modulate the L-type Ca2+ channel (L channel) in vascular smooth muscle cells (VSMC) (10), which play an important role in the maintenance of vascular tone (11, 12, 13).

Alterations in ion channels, including L channels, Ca2+-activated K+ channels, and the voltage-gated outward K+ channels, are involved in the abnormalities observed in the initiation and maintenance of the contractile state of VSMC in hypertension (14). An increase in Ca2+ channel activity plays an important role in the increased contractile tone in VSMC from spontaneously hypertensive rats (SHR), a genetic model of hypertension (15). This increase in Ca2+ channel activity could occur secondary to an increase in channel density and/or altered signaling mechanisms (16, 17, 18, 19, 20).

We recently characterized the effect of PACAP on L channel currents in VSMC prepared from rat tail arteries of Sprague Dawley rats. We found that modulation of L channel currents by PACAP involves activation and integration of two signaling pathways, protein kinase A (PKA), which reduces Ca2+ entry, and protein kinase C (PKC), which promotes Ca2+ entry (10). Therefore, to better understand the vasoactive effect of PACAP and its possible role in blood pressure control, we compared the effects of PACAP on the L channel current in VSMC from SHR and Wistar Kyoto rats (WKY), the normotensive controls. We found that PACAP caused a greater increase in the L channel current amplitude in VSMC from SHR. This increase was probably related in part to the increased L channel current density as well as the enhanced sensitivity to the PKC pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
PACAP-(1–38) was obtained from Peninsula Laboratories, Inc. (Belmont, CA). 4ß-Phorbol 12-myristate 13-acetate (PMA) and 8-bromo-cAMP were obtained from Sigma (St. Louis, MO). Calphostin C was obtained from Calbiochem (San Diego, CA). Adenosine cyclic 3',5'-monophosphothioate, Rp-isomer (Rp-cAMPs) was obtained from BioLog Life Science Institute (La Jolla, CA). All other chemicals were of the purest grades commercially available. [125I]cAMP was obtained from ICN ImmunoBiologicals (Lisle, IL). The antibody for the RIA of cAMP was a gift from Dr. A. Baukal (NICHHD, NIH, Bethesda, MD).

Cell preparation and Ca2+ current recordings
All procedures were reviewed and approved by the health sciences animal and welfare committee of the University of Alberta. Sixteen-week-old male SHR and WKY rats obtained from Taconic Farms (Germantown, NY) were used for this study. Only SHR with established hypertension were used for the cell preparation. VSMC were dispersed enzymatically from at least three SHR and WKY rats using an established method (10). Cells were used within 18–30 h of isolation. Ca2+ channel current recordings were obtained using the whole cell and cell-attached configuration of the patch clamp technique (10, 21). The experiments were performed at room temperature (20-22 C). Patch electrodes were pulled from borosilicate glass capillary tubes (od, 1.2 mm; id, 0.9 mm; FHC, Brunswick, ME) and heat-polished. For the whole cell current, the patch electrodes were filled with a solution containing 70 mM Cs2 aspartate, 20 mM HEPES, 11 mM EGTA, 1 mM CaCl2, 5 mM MgCl2·6H2O, 5 mM glucose, 1 mM ATP-Na2, and 5 mM potassium succinate. Creatine phosphokinase (50 U/ml) and phosphocreatine-Na2 (20 mM) were added to the pipette solution to reduce current rundown. The bath solution contained 105 mM Tris-HCl, 0.8 mM MgCl2, 5.4 mM KCl, 20 mM BaCl2, 0.02 mM tetrodotoxin, and 10 mM HEPES. In all experiments, Ba2+ (20 mM) was used as the charge carrier. The osmolarity was adjusted to 320 mosmol, and the pH to 7.4. All solutions were filtered (0.22 µm) before use.

For single channel current measurements, the patch electrodes were filled with a solution containing 70 mM BaCl2, 10 mM HEPES, and 110 mM sucrose, and pH was adjusted to 7.4 with tetraethylammonium hydroxide. The bath solution contained 120 mM potassium glutamate, 25 mM KCl, 2 mM EGTA, 10 mM HEPES, 2 mM MgCl2, 1 mM Ca2+-ATP, and 10 mM glucose, and pH was adjusted to 7.4 with KOH. The single channel current records were obtained by depolarizing the cell-attached patch from -40 to 20 mV. These were corrected for liquid junction potentials that were 15.3 mV.

The membrane currents were measured using an Axon (Axopatch; ß = 1) patch clamp amplifier (Axon Instruments, Foster City, CA). The data were sampled using pClamp software (pClamp 5.71) and a Digidata 1200 analog to digital interface (Axon Instruments). Analysis was performed using pClamp software (ClampFit8 or Fetchan 8). The effects of the drugs were monitored continuously using depolarizing pulses at a frequency of 0.03 Hz, except when generating I-V relationships. To generate current-voltage (I-V) relationships, 250-msec depolarizing test pulses of increasing amplitude were applied at a frequency of 0.3 Hz. On-line leakage subtraction was implemented using the P/N protocol in pClamp software. In all cases the peak inward current, which represents the maximal inward current over the test voltage range (leak corrected), was used to construct the I-V relationships. Cell capacitance was determined by integrating the area under the capacitative transient after the whole cell voltage clamp was established. Electrode capacitance was subtracted from the total area. The drugs were either dissolved in bath solution or dimethylsulfoxide and added to a static bath. At the maximal concentration of dimethylsulfoxide (0.4%, vol/vol) used, it had no effect on the L channel current. The effects of treatments on the whole cell current were compared using the peak inward current, which usually occurred at 20 mV. To compare the inactivation rates of the whole cell current from WKY and SHR, the peak inward currents (at 20 mV) from 10 cells were averaged and normalized. The single channel measurements were filtered at 2 kHz and sampled at 5 kHz. Current records were corrected for capacitative transients and leak by subtraction of averaged blank sweeps. The data were analyzed using Fetchan and pStat in pClamp6. Open time (dwell time) histograms were obtained from 128 200-msec sweeps. A simplex algorithm in pStat was used to find the maximum likelihood fits. The dwell time histogram was best fitted with two exponentials, a fast open time constant, {tau}f, and a slow open time constant, {tau}s, and percent values are the areas under the curves described by the time constants.

cAMP determination
Dispersed VSMC were plated at a density of 5 x 104 cells/well and maintained in DMEM with FCS (10%, vol/vol) at 37 C. Two days after subculture, the cells were washed once with DMEM containing 0.1% BSA and equilibrated for 1 h in the same medium before performing the experiments. Cellular cAMP content was determined using an RIA procedure in which samples were acetylated before analysis (10, 22).

Data and statistical analysis
Data are presented as the mean ± SEM percentages of control values. Each experiment was repeated at least three times using different cell preparations. The paired t test was used for comparison between control values and those obtained after drug administration. In the case of multiple comparisons, ANOVA in conjunction with the Newman-Keuls test was applied. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of L channel currents in tail artery VSMC from WKY and SHR
VSMC obtained from the tail arteries of SHR and WKY were spindle-shaped and similar in size, ranging from 6–15 µm in width and 100–150 µm in length. Figure 1Go, A and B, shows the inward currents activated in single WKY and SHR VSMC by depolarizing the cells from a holding potential of -40 mV to various test potentials in 20-mV increments. Although there was no difference in the activation rate, the inactivation of L channel current from SHR VSMC was slower ({tau}inactivation WKY, 0.14 ± 0.01 sec; SHR, 0.38 ± 0.10 sec; P < 0.05; Fig. 1CGo). To determine L channel current densities, cell capacitance was used as a measure of surface area (see Materials and Methods). The L channel current density was 41% higher in SHR compared with WKY VSMC (Fig. 1DGo).



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  		Figure 1. Comparison of L channel currents in tail artery VSMC from WKY and SHR. A and B, The inward currents activated in single WKY and SHR VSMC by depolarizing the cells from a holding potential of -40 mV to various test potentials in 20-mV increments. C, The difference in inactivation rates of L channel currents in VSMC from WKY and SHR. The currents shown are the average of 10 peak inward currents from 10 cells with 20 mM Ba2+ as the charge carrier (see Materials and Methods). D, The density of the L channel current in VSMC from 16-week-old WKY and SHR based on peak inward current at 20 mV (n = 5; *, P < 0.05).

 
Single L channel current measurements were performed using the cell-attached configuration. The single channel conductances for both types of cells were similar, 24 pS for SHR and 23 pS for WKY. Figure 2Go, A and B, shows four records indicating the two open states in VSMC from SHR and WKY. The fitting of the open time distributions to two exponentials, a fast component, {tau}f, and a slow component, {tau}s, is shown in Fig. 2Go, C and D. There was no difference in the open time constants in VSMC from SHR and WKY or the percent values for {tau}f and {tau}s (Fig. 2Go, C and D, and inset).



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  		Figure 2. Comparison of the single channel measurements in tail artery VSMC from WKY and SHR. A, Four 200-msec records of a single L channel current in a WKY VSMC. The currents were activated by depolarizing the cell from -40 to 20 mV. The top two records have a few short duration openings, and the bottom two records display long openings. B, Similar records obtained from SHR VSMC. C and D, Dwell time (open time) histograms from the experiments described in A and B. The dwell time histogram shows that the open times could be described by two time constants, a fast time constant ({tau}f) and a slow time constant ({tau}s). The average time constants (n = 5) are plotted in the insets of C and D.

 
To verify that the single channel activity was mediated by L channels, single L channel currents were measured in the presence or absence of BayK 8644 or nifedipine in the pipette solution. In control VSMC from WKY, {tau}s was 3.8 ± 0.1 msec, and {tau}f was 0.5 ± 0.1 msec (n = 3). Inclusion of BayK 8644 (1 µM) in the pipette solution resulted in a {tau}s of 28.9 ± 7.9 msec (n = 3; P < 0.05) and a {tau}f of 1.2 ± 0.5 msec (n = 3). With nifedipine (1 µM) in the pipette solution, there were insufficient openings to plot an open time distribution for the determination of {tau}f and {tau}s. Similar results were obtained in VSMC from SHR (data not shown).

Effect of PACAP on L channel currents
Figure 3Go shows the effect of PACAP on the L channel current in VSMC from SHR and WKY. A holding potential of -40 mV was used to inactivate the T channel current present in tail artery VSMC. Figure 3Go, A and B, shows the effect of PACAP (10 nM) on the combined I-V relationships in five cells. There was no difference in the voltage dependence of L channel currents in VSMC from SHR and WKY. PACAP (10 nM) increased the peak inward current in VSMC from both SHR and WKY. Compared with VSMC from WKY, the percent increase in current by PACAP (1–100 nM) was significantly greater in VSMC from SHR, as shown in Fig. 3CGo.



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  		Figure 3. Effect of PACAP on the L channel current in tail artery VSMC from WKY and SHR. A and B, The effect of PACAP (10 nM) on the I-V relationship in VSMC from WKY and SHR (n = 5). C, The effect of PACAP as a function of concentration on the L channel currents in VSMC from WKY and SHR. The data were plotted as a percentage of the control peak inward current at 20 mV before PACAP. *, P < 0.05, significantly different between WKY and SHR (n = 5 for each concentration of PACAP).

 
Figure 4AGo shows the effect of PACAP on single L channel current measurements (cell-attached configuration) in VSMC from SHR and WKY. PACAP (10 nM) caused a greater increase in the ensemble-averaged current, a measurement that is independent of changes in current density, in VSMC from SHR compared with WKY (Fig. 4AGo). Although PACAP (10 nM) had no effect on {tau}f, it caused a significant increase in {tau}s in VSMC from both WKY and SHR (Fig. 4BGo), suggesting that PACAP also had an effect on channel gating that was characterized by a longer mean open time. However, the effect of PACAP on {tau}s in VSMC from SHR was 3-fold higher than that in VSMC from WKY (Fig. 4BGo). The percent value for {tau}s (the area under the curve described by {tau}s) was also increased by PACAP in SHR VSMC from 11.0 ± 2.6% to 21.0 ± 4.9%.



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  		Figure 4. Comparison of the effect of PACAP on the single channel measurements in tail artery VSMC from WKY and SHR. A, Five 200-msec records of single L channel currents in WKY and SHR VSMC before and after PACAP (10 nM). L Channels were activated by depolarizing the cells from -40 to 20 mV. Shown below the current records are ensemble-averaged currents from 64 200-msec sweeps. Open time distributions were obtained before and after PACAP (10 nM) from 128 200-msec sweeps. The open time distributions were fitted to 2 exponentials, {tau}f and {tau}s, as described in Fig. 2Go, C and D. The time constants, {tau}f and {tau}s, from several cells were averaged and plotted as shown in B.

 
Role of PKC to the enhanced PACAP response in VSMC from SHR
PACAP is known to activate PKC and PKA in VSMC from Sprague Dawley rats, with PKC increasing and PKA reducing the L channel current (10). Therefore, the enhanced PACAP response in VSMC from SHR could be secondary to an increase in PKC sensitivity. This was examined using PMA, an activator of PKC. Figure 5Go, A and B, shows the effect of PMA (500 nM) on the combined I-V relationships in five cells. PMA increased the peak inward current in VSMC from both SHR and WKY, with the increase significantly greater in VSMC from SHR at concentrations between 50 and 500 nM (Fig. 5CGo). Furthermore, PMA appeared to shift the peak of the I-V relationship toward more negative potentials by about 5 mV in SHR VSMC, an effect not observed in WKY (Fig. 5Go, A and B).



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  		Figure 5. Effect of PMA, a PKC activator, on the L channel current in VSMC from WKY and SHR. A and B, The effect of PMA (500 nM) on the I-V relationship in VSMC from WKY and SHR (n = 5). C, The effect of PMA as a function of concentration on L channel currents in VSMC from WKY and SHR. The data were plotted as a percentage of the control peak inward current at 20 mV before PMA. *, P < 0.05, significantly different between WKY and SHR (n = 5 for each concentration of PMA).

 
Figure 6AGo shows five records of single L channel current measurements (cell-attached configuration) before and after PMA in VSMC from SHR and WKY. PMA (500 nM) caused a greater increase in the ensemble-averaged current in VSMC from SHR than in those from WKY (Fig. 6AGo). PMA (500 nM), like PACAP, had no effect on {tau}f, but caused a significant increase in {tau}s in VSMC from both WKY and SHR (Fig. 6BGo). However, the effect of PMA on {tau}s in VSMC from SHR was 3.5-fold higher than that from WKY (Fig. 6BGo). PMA also increased the percent value for {tau}s in SHR from 9.6 ± 2.9% to 29.0 ± 4.1%.



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  		Figure 6. Comparison of the effect of PMA, a PKC activator, on open times in WKY and SHR VSMC. A, Five 200-msec records of single L channel currents in WKY and SHR VSMC before and after PMA (500 nM). Shown below the current records are ensemble-averaged currents from 64 200-msec sweeps. B, Summary of the data on open time distributions from 128 200-msec sweeps before and after PMA (n = 4). The time constants were obtained as described in Fig. 2Go, C and D, and are plotted as a histogram.

 
Role of PKA on the enhanced PACAP response in VSMC from SHR
The enhanced PACAP response in VSMC from SHR could also be secondary to a reduced sensitivity to PKA. This was examined using 8-bromo-cAMP, a membrane-permeable cAMP analog. 8-Bromo-cAMP (100 µM) caused a similar reduction in the peak inward current in VSMC from SHR and WKY (Fig. 7AGo).



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  		Figure 7. Role of cAMP on the PACAP effect on the L channel current in rat tail artery VSMC from WKY and SHR. A, The effect of 8-bromo-cAMP (8 Br-cAMP; 100 µM) on the L channel current in VSMC from SHR and WKY (n = 4). The data were plotted as a percentage of the control peak inward current at 20 mV before 8 Br-cAMP. There was no difference between the effect of 8 Br-cAMP on VSMC from SHR and WKY. B, The effect of PACAP (10 nM) on cAMP accumulation in VSMC from SHR and WKY. At the end of 15 min, cells were pelleted, and cAMP was determined by RIA. Each value represents the mean ± SEM of determinations performed in quadruplicate from three independent experiments. *, P < 0.05, significant difference between WKY and SHR.

 
To determine whether cAMP production was reduced in VSMC from SHR, cells were treated with PACAP (10 nM) for 15 min. PACAP caused an increase in cAMP accumulation in VSMC from SHR and WKY (Fig. 7BGo). PACAP-stimulated cAMP production was reduced in VSMC from SHR (Fig. 7BGo).

To determine the contributions of PKC and PKA to the altered PACAP response in VSMC from SHR, cells were pretreated with Rp-cAMPs, a cAMP antagonist. Rp-cAMPs (100 µM) was effective in blocking the 8-bromo-cAMP (100 µM)-mediated decrease in L-channel current (Fig. 8Go, inset). In the presence of Rp-cAMPs (100 µM), PACAP (10 nM) remained effective in increasing the L channel current in both SHR and WKY VSMC, with a significantly greater response in SHR (Fig. 8Go). However, when the PKC pathway was inhibited by calphostin C (300 nM), PACAP (10 nM) caused a similar reduction of the L channel current in SHR and WKY VSMC (Fig. 8Go). Therefore, the PKA pathway does not appear to play an important role in the increased L channel response to PACAP in VSMC from SHR.



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  		Figure 8. Comparison of the effects of PKA and PKC inhibitors on the whole cell current before and after PACAP in tail artery VSMC from WKY and SHR. The effect of PACAP (10 nM) in the presence of Rp-cAMPs and calphostin C is shown. The data were plotted as a percentage of the control peak inward current at 20 mV before drug treatments. *, P < 0.05, significant difference between WKY and SHR (n = 5). Rp-cAMPs (100 µM) had no effect on the L channel current of VSMC from WKY and SHR, and calphostin C (300 nM) caused a small decrease in the L channel current in VSMC from WKY and SHR (data not shown). Shown in the inset is the effect of 8-bromo-cAMP (8 Br; 100 µM) on the L channel current in VSMC from WKY in the presence or absence of Rp-cAMPs (Rp; 100 µM; n = 4). Rp-cAMPs blocked the 8 Br-mediated decrease in the L channel current.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Rat tail arteries are resistance vessels that specialize in thermogenesis and have been used extensively in the study of the pathogenesis of hypertension (23, 24). It has previously been shown that an alteration in Ca2+ channel function and/or an increase in Ca2+ channel density underlies the increased vascular reactivity characteristic of hypertension (14, 15, 16, 17, 18, 19, 20). However, there has been no consensus on the cellular mechanisms involved. In this study using PACAP, an endogenous ligand with vasoactive properties that activates two important signaling mechanisms that regulate L channels, PKC and PKA, we found that PACAP caused a greater increase in the amplitude of the L channel current in VSMC from SHR at an age in which hypertension is established. Furthermore, the L channel current density is increased in VSMC from SHR.

Regardless of the source of the arteries, a higher L channel current density in myocytes appears to be a common finding in SHR compared with WKY (17, 18, 19, 20). In the present study we found a 40% higher L channel current density in VSMC prepared from tail arteries of 16-week-old SHR, in agreement with a recent study that found a persistent increase in current density in myocytes from 5- to 23-week-old animals (25). However, it should be mentioned that in an earlier study the increase in current density was found to be age dependent and was only observed in 5-week-old, not in 16- to 18-week-old, animals (18). With respect to single channel characteristics, although we did not find any difference in single channel conductance or open time between SHR and WKY VSMC, a previous study reported a difference in open frequency (26). Potentially, this difference could be related to the source of VSMC and/or the difference in the subunit composition of the channel, issues that warrant additional investigation.

In addition to an increase in L channel current density in VSMC from SHR, our study shows a difference in the PACAP response in VSMC from SHR and WKY. PACAP, apart from causing an increase in the amplitude of the whole cell current, also changes gating of the channel in VSMC from SHR and WKY. Similar to other studies, activation of L channels leads to two open states, a fast component, {tau}f, and a slow component, {tau}s (27). PACAP appears to selectively increase the {tau}s of the open times without having an effect on the {tau}f in both SHR and WKY VSMC. Moreover, PACAP causes a greater increase in the {tau}s in SHR VSMC compared with that in WKY. An increase in {tau}s is due to a change in channel gating characterized by a longer mean open time. Whereas the increase in L channel current density in VSMC from SHR could explain in part the PACAP-mediated increase in amplitude of the whole cell current, it could not explain the greater increase in {tau}s and the ensemble-averaged current after PACAP, which are independent of changes in current density. Therefore, our results suggest that in addition to changes in current density, an alteration in Ca2+ channel regulation may be equally important in contributing to the PACAP response in SHR.

Our results further suggest that the greater PACAP effect on the L channel VSMC from SHR is probably due to an enhanced PKC response. This is based on the observations that treatment with PMA, like PACAP, also increases the amplitude of the L channel current and prolongs the open times that are characterized by an increase in {tau}s in VSMC from SHR and WKY. In addition, there is a greater increase in the PMA-induced increase in amplitude of the L channel current, {tau}s, of the open time and the ensemble-averaged current in SHR VSMC compared with WKY. Furthermore, by using selective inhibitors of the PKA and PKC pathways, our results support an increased PKC response in SHR VSMC. In the presence of a PKC inhibitor, there is no difference in the effect of PACAP on the L channel current between SHR and WKY VSMC. Consistent with our findings, previous studies in hypertensive animals have shown an increase in phospholipase C activity or PKC response (28, 29, 30, 31).

Like the contribution of the PKA pathway, 8-bromo-cAMP causes a reduction of the L channel current amplitude in VSMC from SHR and WKY. However, treatment with PACAP causes a smaller increase in cAMP production in SHR myocytes. Therefore, it is possible that the reduced cAMP production may amplify the effect of the PKC pathway on the L channel current. Because the difference in the L channel response to PACAP between SHR and WKY VSMC persists in the presence of Rp-cAMPs, a cAMP antagonist, this argues against a major contribution of the PKA pathway to this difference.

In summary, SHR VSMC prepared from rat tail arteries have a higher L channel current density compared with those from WKY. PACAP, in addition to increasing the amplitude of the L channel current, affects L channel gating, characterized by a longer mean open time in SHR and WKY VSMC. Furthermore, PACAP evokes a greater response in SHR ensemble-averaged currents than in WKY, a measurement that is independent of current density. Moreover, we found that changes in the sensitivity of the PKC, not the PKA, pathway appear to play a dominant role in the difference in PACAP response between SHR and WKY VSMC. Our results are of interest because they provide a cellular mechanism that could explain the enhanced effect of PACAP on the L channel current in VSMC from SHR. The enhanced sensitivity of the PKC pathway in SHR VSMC could represent an important mechanism that mediates the vascular hyperreactivity observed in hypertension. Because we only used SHR with established hypertension, we could not exclude the possibility that the enhanced PKC sensitivity is secondary to the state of hypertension.


   Footnotes
 
1 This work was supported by grants from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada. Back

2 B.L. and C.L.C. are equal first authors. Back

Received November 30, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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