(Hypertension. 1996;28:772-778.)
© 1996 American Heart Association, Inc.
Articles |
Angiotensin II–Stimulated Phospholipase C Responses of Two Vascular Smooth Muscle–Derived Cell Lines
Role of Cyclic GMP
the Departments of Cell Physiology and Pharmacology and of Medicine and Therapeutics (L.L.N.), School of Medicine, University of Leicester (UK).
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Abstract |
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Vascular smooth muscle cells of the spontaneously hypertensive rat (SHR) are known to show increased responsiveness to angiotensin II (Ang II) compared with cells of normotensive control Wistar-Kyoto rats (WKY). We investigated the hypothesis that differential levels of cGMP lead to the different responsiveness of the cells, using vascular smooth muscle cells in culture. cGMP levels in extracts of SHR-derived cells were lower than those of WKY-derived cells. This was true for both unstimulated cells and cells treated with equal concentrations of either sodium nitroprusside or S-nitroso-N-acetylpenicillamine. Stimulation of cells with Ang II did not affect levels of cGMP but increased levels of inositol 1,4,5-trisphosphate (IP3) and Ca2+, which were greater in SHR- than in WKY-derived cells. When SHR and WKY cells were preincubated with different concentrations of S-nitroso-N-acetylpenicillamine to generate similar cGMP levels in each cell type, the subsequent IP3 response to Ang II was the same in the two cell types. To reduce any influence of cGMP on responses, we permeabilized the cells with
-toxin. Stimulation of -toxin–permeabilized cells with high Ca2+ revealed an IP3 response in SHR- but not WKY-derived cells. Similarly, permeabilized SHR cells responded to Ang II but WKY cells did not. However, GTP and GTP
S elevated IP3 in both cell types. Taken together, these results indicate that the low response of WKY cells can be accounted for by the inhibitory influence of cGMP. However, when this inhibition is removed by permeabilization, further differences between the cells are revealed that will contribute to the elevated SHR response.
Key Words: muscle, smooth vascular • rats, inbred SHR • angiotensin II • cyclic GMP • phospholipase C
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Introduction |
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The mechanism whereby vasoconstrictor substances (such as Ang II and endothelin) influence VSM cells is poorly understood. Ang II acts on a seven-transmembrane G protein–linked receptor to stimulate phosphoinositide-specific PLC, which controls intracellular Ca2+ and protein kinase cascades by the formation of IP3 and diacylglycerol. There is now a well-established body of literature describing the regulation of PLC isoforms (see Reference 1 for review). The PLCß isoforms are required for G protein responses (by both
GTP- and ß-subunits); PLC isoforms are regulated by association with (and phosphorylation by) activated tyrosine kinase growth factor receptors; and PLC
regulation is unclear but may be downstream of elevated Ca2+. In VSM cells PLCß is reported to be lacking,2 3 4 whereas PLC and PLC are present. It is possible that functional amounts of certain PLCß isoforms exist that are linked to G proteins. Alternatively, it has been shown that Ang II causes PLC phosphorylation and activation in these cells,4 although the mechanism by which this occurs is unclear.
The SHR exhibits several features of interest in this respect compared with normotensive controls such as the WKY. SHR vasculature has been shown to give enhanced responses to Ang II and other agonists acting on G protein–linked receptors.5 6 7 8 9 Despite some conflicting evidence,6 the majority of studies show that SHR-derived VSM cells do not have a higher number of Ang II receptors.7 10 SHR-derived VSM cells have been shown to differ with respect to greater responsivity of the PLC isoform.2 This would be expected to contribute to the enhanced responses of the SHR-derived tissue. A model can be proposed in which the receptor stimulates PLC or PLCß (see above), which produces a priming degree of Ca2+ stimulation; this activates PLC, which then acts as an amplifier in which the gain is greater for SHR-derived cells than for WKY-derived cells.
In addition, however, there are indications that cGMP may play a role in the differential responsiveness of VSM cells from the two rat strains. It is well established that the endothelium regulates VSM function by the release of both vasodilator and vasoconstrictor substances. NO is a vasodilator that stimulates soluble guanylate cyclase; the consequent elevation in cGMP can exert a vasodilator effect at several levels of VSM function, including inhibition of phosphatidylinositol hydrolysis in the aorta.11 12 13 Evidence has been presented that the vasodilator influence of the endothelium, and in particular the contribution of NO, is deficient in the SHR model of hypertensive disease.14 15 16 17 18 19
In preliminary studies, we noted that cultured VSM cells of WKY origin had higher levels of cGMP than the equivalent cells of SHR origin. In the present study, we have characterized the difference in the PLC responses of the two cell lines to stimulation with Ang II and investigated the role of cGMP in generating this difference. We provide data suggesting that enhanced responsiveness of the SHR-derived cells is due to an attenuated cGMP influence superimposed on the differential contribution of PLC as indicated in the model set out above.
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Methods |
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Materials
SHR and WKY were from the colonies of the Biomedical Services Unit of Leicester (UK) University. Cell culture supplies were from GIBCO, Ang II and fura 2 from Sigma Chemical Co, and radiochemicals from Amersham. Staphylococcus
-toxin was a kind gift of Dr Kati Torok, Department of Physiology, University College London (UK).
Cell Culture
Cells were prepared as described in Reference 20. Arterial pressure of 12-week-old SHR and WKY was determined, and cells were digested from 1-mm pieces of thoracic aorta. Clonal colonies with smooth muscle morphology were combined for generation of SHR- and WKY-derived cell lines with 100% positive smooth muscle actin immunofluorescence that were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 27 mg/mL glutamine. Cells were grown close to confluence in 24-well plates and were used between passages 6 and 12.
Measurement of IP3 Levels
Cells were washed free of growth medium with balanced salt solution (BSS) comprising (mmol/L) NaCl 125, KCl 5.4, NaHCO3 16.2, HEPES 30, NaH2PO4 1, MgSO4 0.8, CaCl2 1.8, and glucose 5.5 buffered to pH 7.4 with NaOH and gassed with 95% O2/5% CO2. Incubations for the times indicated were with agonist in 200 µL BSS and were stopped with 100 µL of 1.5 mmol/L trichloroacetic acid. After removal of the acid with ether washes, the extracts were assayed for the mass of IP3 with a protein binding procedure.21 The protein content of each batch of cells was determined at the time of use. Data are expressed per microgram of protein.
Measurement of Free Intracellular Ca2+
Cells that had just reached confluence on 9x22-mm glass coverslips were loaded with fura 2-AM for 1 hour at 37°C in medium M199 buffered to pH 7.4 and supplemented with 10 mmol/L HEPES and 0.1% bovine serum albumin. After deesterification for 30 minutes, fluorescence was measured in 1.8 mmol/L CaCl2, 0.8 mmol/L MgSO4, 15 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L KCl, and 5 mmol/L glucose, buffered to pH 7.4, in a dual excitation fluorometer (Deltascan, Photon Technology International) recording ratios of excitation at 340/380 nm with emission at 530 nm. Ratios were calculated after correction of autofluorescence. Calibrations were performed by permeabilizing cells with the nonfluorescent calcium ionophore 4-bromo-A23187 and measuring the 340/380 nm Rmin and Rmax values in buffers containing EGTA (1 mmol/L, pH 8.5) or calcium (2 mmol/L). Data are presented as this ratio and also as the change in the calculated calcium concentrations, as described previously.22
Measurement of cGMP Levels
Cells on multiwell plates were washed twice with 1 mL BSS supplemented with 300 µmol/L IBMX unless otherwise indicated. After 20 minutes, drugs were added in 0.5 mL BSS with 300 µmol/L IBMX. The reaction was stopped by aspiration and addition of 0.5 mL cold trichloroacetic acid (0.5 mol/L). After removal of acid by ether washing, the samples were assayed for cGMP by radioimmunoassay.23 Data are presented as femtomoles per microgram of protein.
Permeabilization Procedure
Cells in 24-well plates were washed and permeabilized by incubation for 20 minutes with -toxin at 50 U/mL in 200 µL of the following intracellular buffer (mmol/L): HEPES 15, potassium gluconate 140, magnesium acetate 7, glucose 5, EGTA 1, and ATP 5. The Ca2+ concentration was buffered to 80 to 120 nmol/L (with EGTA, calibrated with quin 2), and bovine serum albumin was added to 0.1%. The permeabilizing medium was then removed and replaced with 200 µL of the same buffer containing Ang II, GTP, GTPS, or Ca2+. The reaction was terminated after 15 seconds by the addition of 21 µL trichloroacetic acid (5 mol/L), and IP3 was measured by a protein binding assay as above.
Miscellaneous
Protein was determined by the procedure of Lowry et al,24 and significance was tested with the two-tailed Student's t test. Curve fitting was performed with a recursive nonlinear least-squares algorithm (P-fit, Biosoft Corp).
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Results |
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cGMP in Cells Derived From WKY and SHR
In preliminary experiments, cGMP levels measured in WKY and SHR cell extracts from first-passage cultures showed about a twofold difference, WKY measurements being significantly higher (P<.002, n=14). This difference between the cell types at the first passage was sustained and more pronounced at later passages (passages 6 to 12). The pattern of this difference between the SHR and WKY cells, with and without stimulation by SNAP, is illustrated in Fig 1
, for which later-passage cells were used. These results also show that the differences in cGMP levels in the SNAP-stimulated cells were observed even in the absence of IBMX. Since the supply of these later-passage cells was not limiting, these were used subsequently for the main body of the work. The EC50 for stimulation of cGMP levels by SNAP in the SHR cells was smaller than that for the WKY cells (pooled across three separate experiments, 0.249±0.067 versus 0.463±0.066 µmol/L). This difference in EC50 of the two cell types for SNAP may reflect a qualitative difference in the predominant form of soluble guanylate cyclase.
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PLC Responses
The responses measured downstream of PLC stimulation were the immediate product of hydrolysis of phosphatidyl 4,5-bisphosphate, the Ca2+ mobilizing IP3, and the Ca2+ response itself. Fig 2 shows the IP3 responses to 100 nmol/L Ang II for each cell type. In the SHR cells, there is a clear peak at 15 to 20 seconds, which then forms a plateau for the duration of the experiment (5 minutes). By contrast, the WKY cells generated a much smaller peak, which returned to basal within 1 minute; there was no plateau. When a shorter time course of stimulation was undertaken, a biphasic time course was seen, with an initial phase complete at 5 to 7 seconds followed by a second phase that peaked at 15 seconds. Both phases were higher in SHR than in WKY cells (data not shown).
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Resting cytosolic Ca2+ concentrations in SHR and WKY cells were 121±9.4 and 107±7.3 nmol/L, respectively (n=18, P=NS). Fig 3 shows a series of measurements of the cytosolic Ca2+ response to different Ang II concentrations. The higher Ang II concentrations (1000 nmol/L, Fig 3A) generated a peak and plateau in the SHR cells and a smaller but discernible peak and plateau in the WKY cells. The WKY response was smaller in both peak and plateau in every experiment. With 3 nmol/L Ang II (Fig 3B), the SHR response was more rapid, reaching a maximum at about 20 seconds, whereas the WKY response was smaller and slower, being similar to the SHR response to 1 nmol/L Ang II. At 1 nmol/L Ang II, WKY cells did not respond (Fig 3C). SHR cells showed a strong response that consisted of a gradual increase in intracellular Ca2+ to a maximum at about 90 seconds. These differences were also evident with data pooled across experiments, as shown in Fig 4, which indicates the dose-response curves for Ang II with the peak (Fig 4A) and plateau (Fig 4B) for the two cell types. These differences were not due to differences in fura 2 loading leading to variation in intracellular Ca2+ buffering because equal numbers of cells loaded with fura 2 on the coverslips emitted similar levels of fluorescence at the isosbestic wavelength of 360 nm. Furthermore, the Rmax, Rmin, and fluorescence emisson ratio (Sf/Sb) at 380 nm for Ca2+-free and Ca2+-saturated forms of fura 2 measured in the presence of 4-bromo-A23187 in SHR and WKY cells were not significantly different, indicating that the fluorescent properties of the intracellular dye in the two cell types were similar (SHR cells: Rmax, 4.31±0.82; Rmin, 0.35±0.03; Sf/Sb, 3.92±0.50; WKY cells: Rmax, 4.07±1.10; Rmin, 0.34±0.03; Sf/Sb, 3.50±0.70).
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In further experiments, we showed that removal of extracellular Ca2+ caused a reduction in the size of the peak response to 1000 nmol/L Ang II but left most of it intact, while the plateau largely disappeared (data not shown). This was consistent with the peak being dominated by Ca2+ release from intracellular stores and the plateau being mainly sustained by Ca2+ entry from the extracellular compartment.
Interaction of cGMP With the PLC Responses
For investigation of whether the difference in cGMP between the two cell types could be contributing to the difference in PLC responses, it was necessary to demonstrate that the elevation of cGMP in the SHR cells would reduce this response. This is shown in Fig 5 for the Ca2+ response and in the Table for the IP3 response. Both Fig 5 and the Table illustrate that the SHR cells responded to Ang II in a manner that was inhibited but not eliminated by SNAP. Pooled across experiments, the peak Ca2+ incremental response (reported as 340/380 nm ratio and as cytosolic Ca2+ concentrations within parentheses) to 300 nmol/L Ang II was reduced by 100 µmol/L SNAP from 0.865±0.133 (330±64 nmol/L, n=7) to 0.454±0.084 (148±31 nmol/L, n=4; P<.05), and the plateau Ca2+ incremental response was reduced from 0.447±0.045 (145±17 nmol/L, n=7) to 0.244±0.041 (73±13 nmol/L, n=4; P<.01).
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We undertook a series of experiments designed to investigate whether the synthesis of NO by NO synthase in the cells generates the difference in cGMP and whether Ang II affects the level of cGMP. The results showed that neither the NO synthase inhibitor L-NMMA nor Ang II altered cGMP levels in either cell type. For example, incubation of WKY cells for 20 minutes with 100 nmol/L L-NMMA gave 2.92±0.19 fmol cGMP/µg protein compared with a value in the absence of L-NMMA of 2.76±2.84 fmol/µg (n=3). Concentration-response studies confirmed that Ang II failed to affect cGMP levels between 1 and 1000 nmol/L. For example, 20 minutes of stimulation of SHR cells with Ang II (1000 nmol/L) gave 0.89±0.16 fmol cGMP/µg protein compared with an unstimulated control value of 0.88±0.01 fmol/µg (n=3). Further experiments showed that L-NMMA did not affect the concentration-response curve for Ang II stimulation of IP3 in both the WKY and SHR cells and that methylene blue, an inhibitor of soluble guanylate cyclase, failed to alter the Ca2+ response to Ang II in the WKY cells (data not shown). Furthermore, methylene blue failed to alter cGMP levels in either cell type (methylene blue compared with control cGMP levels in SHR, 92±10%; in WKY, 108±11%). These experiments led us to conclude that under the culture conditions used here, NO-stimulated guanylate cyclase was not involved in generating the differential levels of cGMP nor in the differential PLC responses between the cells.
Nevertheless, it remained possible that the lowered responsiveness of the WKY cells was due to the higher cGMP levels and that when stimulated with NO (or SNAP), the resultant elevation in cGMP would contribute to the differential responses to Ang II. To directly test this hypothesis, we treated the WKY and SHR cells with different SNAP concentrations that gave rise to similar cGMP levels in the cells (100 µmol/L SNAP for SHR cells and 0.1 µmol/L for WKY cells). This was confirmed by measurement of cGMP in the SNAP-treated cells, which demonstrated that WKY cGMP levels were no longer significantly different from those of SHR cells, being 94±8% of the SHR cGMP levels. Having achieved approximate equilibration of cGMP levels, we then stimulated the cells with Ang II and measured the IP3 response. Under these conditions, the responses of WKY and SHR cells did not differ (Table
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Stimulation of Staphylococcus -Toxin–Permeabilized Cells
On permeabilization with -toxin, cGMP levels fell in both cell types. In extracts from both cell types, the levels fell below the detectable limits for our cGMP assay. Not only was the cGMP level drastically reduced, but figures for residual cGMP showed that it was not different between the two cell types (SHR, 0.059±0.006 fmol/µg protein; WKY, 0.069±0.007, n=8; not significantly different from each other but significantly different from untreated cells, P<.001 in each case). Under these conditions, the PLC responses of G protein–linked receptors are sustained, and the SHR and WKY responses can be compared free of the effects of cGMP. In Fig 6 it can be seen that both cell types retained their responsiveness to GTPS, generating an accumulation of IP3. This shows that both cell types retained the G protein–PLC link after permeabilization. When the cells were stimulated with 1 µmol/L Ca2+, there was a substantial increase in the SHR cells but no increase in the WKY cells. When SHR cells were stimulated with Ang II, there was a severalfold response with the agonist alone and a larger response in the simultaneous presence of GTP or GTPS; with WKY cells, there was no response to Ang II under any of these conditions.
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Discussion |
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It has been demonstrated that cultures of VSM cells from WKY and SHR retain many differences between them through several passages. Over a series of experiments, we have shown that after 6 to 12 passages, the difference in basal and SNAP-stimulated cGMP levels between WKY and SHR cells is retained. We have also shown that the PLC response to Ang II in the SHR-derived cells is higher than in the WKY-derived cells. We then investigated the relationship between this elevated cGMP and the diminished response to Ang II in the WKY cells.
Major determinants of VSM cGMP levels include (1) the production of cGMP from stimulation of soluble guanylate cyclase by NO and of particulate guanylate cyclase by atrial natriuretic factor and (2) the loss of cGMP from cells because of its breakdown by phosphodiesterases or egression to the extracellular space. NO is a vasodilator, and its deficiency has previously been implicated in the maintenance of hypertension in the SHR model,17 18 19 part of a larger body of evidence indicating the significance of deficient endothelial vasodilator influence. However, the role of NO in this deficient endothelial influence can be questioned, with the demonstration of a contribution by endothelium-derived hyperpolarizing19 25 and contracting26 27 factors. Furthermore, it has been shown that particulate guanylate cyclase is likely to contribute to differences in VSM cGMP levels.28 The current observations that SNAP-stimulated cGMP levels are higher in WKY than in SHR cells indicate that soluble guanylate cyclase plays a role in the differential cGMP levels in cells subject to the influence of endothelium-derived NO. We then investigated whether the differential PLC response to Ang II is a result of the difference in cGMP levels.
It is noteworthy that in neither of the PLC-related responses studied (IP3 and cytosolic Ca2+ levels) was there a difference in basal levels between the two cell types. That this reflects the in vivo situation is suggested by studies on endothelium-denuded mesenteric resistance arteries, in which basal Ca2+ did not differ between SHR and WKY,29 and on endothelium-denuded aortic rings and tail arteries, in which no difference in basal inositol phosphates between the two rat strains was found.30 31 By contrast, there are reports of increased Ca2+ levels in unstimulated explanted blood vessels from SHR.31 32 It is also instructive to compare our results on stimulated cultured cells with agonist-stimulated blood vessels acutely removed from SHR and WKY. Strict comparison is made difficult by the variety of blood vessels chosen and of agonists used, so it is therefore not surprising that no clear pattern emerges. For example, aortic inositol phosphate responses to 5-hydroxytryptamine have been reported to be higher in SHR than in WKY cells,33 whereas the 1-adrenoceptor inositol phosphate responses in some vessels from SHR are lower than in vessels from normotensive controls.34 Aortic rings derived from adult SHR have been shown to give a smaller response than the WKY equivalent to stimulation with endothelin,30 and total inositol phosphate responses to norepinephrine in tail artery do not differ between the two rat strains.31 Complexities in the interpretation of some of these data may relate to the analytic methods used. This is illustrated by a report indicating that with norepinephrine-stimulated tail arteries, the total inositol phosphate response is the same in the two rat strains but that this reflects an increased IP3 response and reduced inositol monophosphate response in the SHR cells.35
In the present report, we avoided this type of confusion by using an assay for the mass of IP3, which is the inositol trisphosphate isomer that is the primary product of the PLC reaction and is also the isomer responsible for controlling intracellular Ca2+. We produce simply interpretable results with cultured cells which show that in SHR cells, there is an increased peak and plateau response to Ang II stimulation with both IP3 and Ca2+ measurements. Such findings were not due to altered buffering of the intracellular Ca2+ by differences in fura 2 loading or to variation in the intracellular fluorescent properties of the fluorophore. These results thus reflect a reduction in WKY-derived cells in the stimulation of PLC, accumulation of IP3, mobilization of intracellular Ca2+, and entry of Ca2+ from the extracellular compartment.
Several observations indicate that the inhibitory influence of cGMP on PLC responses is involved in this reduced response, including our own finding of a difference in cGMP levels between the two cells and several reports on vasculature that cGMP can inhibit stimulated PLC.11 12 We have partially characterized the difference in cGMP. The basal and nitrosovasodilator-stimulated levels are both higher in WKY cells, whereas neither cell type responds to Ang II with a change in cGMP. The elevated levels in WKY cells are not reduced by L-NMMA and therefore are not due to enhanced NO synthase activities in the cultures. Such differences between the SHR and WKY cells in basal and nitrosovasodilator-stimulated cGMP levels are present in the absence and presence of IBMX and are thus unlikely to be due to differences in the breakdown rates of cGMP. Thus, these differences between cell lines are likely to reflect differences in cGMP production rates. The nitrosovasodilators show a reduced potency (despite a higher maximal response) in the WKY cells. If this pertains in vivo, it may mean that the vasodilator effects of high levels of endothelial NO release are amplified in the WKY, whereas the vasodilatation in response to low levels of NO release is not so different.
Two basic observations in the present study are that the cGMP levels are higher in the WKY cells and that the higher PLC responses of the SHR cells can be attenuated by raising the cGMP levels. This led us to the experiment in which we manipulated cGMP levels by different SNAP concentrations so that levels were the same in SHR and WKY cells. Under these conditions, the PLC response to Ang II was the same in the two cell types. This shows that the differential responsiveness of WKY and SHR cells can be partly accounted for by the difference in cGMP levels.
However, there were reasons to consider the situation beyond this experimental design. First, the different SNAP concentrations used could have other effects on the cells; non-cGMP effects of high concentrations have been described, eg, the reversible inhibition of protein kinase C.36 Furthermore, a previous report indicated that the two cell types differed fundamentally in the nature of the PLC present2 ; therefore, there were likely to be other differences between the cells not revealed by this experimental approach of elevating cGMP to similar levels in the two cell types. We therefore permeabilized the cells before stimulation and washed them, thereby reducing the cGMP levels in the two cell types to the limits of detectability. Permeabilization with -toxin was chosen because it does not permit the loss of proteins, including those of the size of PLC, the smallest of the PLC isoforms and reported to be the form of activity that is enhanced in SHR cells.2 The mode of PLC activation is unclear but may be by elevation of Ca2+.1 Stimulation of the permeabilized cells with high Ca2+ is consistent with this because we show here a substantial response to 1 µmol/L Ca2+ in the SHR but not the WKY cells. The suggestion that PLC can be activated by G proteins, perhaps by interaction of ß-subunits with its pleckstrin homology domains, has also emerged.37 Although this is not supported by the main body of studies on activation of PLC isoforms, it must be considered in view of a recent report suggesting G protein activation of PLC in cells with high expression of the recombinant protein.38 The results reported here showing that GTPS, which will constitutively activate heterotrimeric G proteins, gives a similar response in permeabilized cells of the two types may be a result of stimulation of equal, priming amounts of PLCß, and the differential response of PLC to Ca2+ is not recruited in these permeabilized cells. However, the IP3 response to Ca2+ could be due to activation of PLC, but PLC is activated by G proteins equally in both cell types because the form present in the two cell types differs only in its activation by Ca2+, not in its activation by G proteins.2 The central observation with the permeabilized cells is the retention of responses to Ang II in the SHR cells but not in the WKY cells, indicating that the cGMP levels are not the only determinant of the difference between these cells. The observation of different responses in the permeabilized cells, with both Ca2+ and Ang II giving a response only in the SHR cells, is consistent with the earlier report of a fundamental difference in PLC between the cell types.2 The mechanism is perhaps most likely to be the local production of high Ca2+ by priming amounts of PLC in the permeabilized cells, which is amplified into a measurable response by PLC in the SHR cells but not the WKY cells. Testing this hypothesis will require further studies on Ca2+ mobilization in the permeabilized preparation.
The present results lead to the following clear conclusions: (1) The cGMP concentration in WKY cells is higher than in SHR cells, but this difference is not maintained by NO-sensitive soluble guanylate cyclase. (2) However, under conditions of stimulation with NO donors, this guanylate cyclase does contribute to the difference between the cells. (3) When the cGMP concentration in the two cell types is raised to approximately the same level, just above that in unstimulated WKY cells, the difference in PLC stimulation by Ang II is lost. (4) However, when cGMP is lowered in the two cell types, there is a much greater responsiveness of SHR cells. Under the standard conditions of our cultured cells, this indicates that the cGMP-dependent inhibition of WKY cells and the intrinsic hyperresponsive PLC in the SHR cells both contribute to the difference between the cells.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was funded by The Wellcome Trust.
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Footnotes |
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Reprint requests to Dr L.L. Ng, Division of Clinical Pharmacology, Department of Medicine and Therapeutics, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK.
Received March 26, 1996; first decision April 24, 1996; accepted May 31, 1996.
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References |
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1. Rhee SG, Choi KD. Regulation of inositol phospholipid-specific phospholipase C isozymes. J Biol Chem. 1992;267:12393-12396.
2.
Kato T, Fukami K, Shibasaki F, Homma Y, Takenawa T. Enhancement of phospholipase C activity in the aorta of spontaneously hypertensive rats. J Biol Chem. 1992;267:6483-6487.
3.
Homma Y, Sakamoto H, Tsunoda M. Aoki M, Takenawa T, Ooyama T. Evidence for early involvement of phospholipase C-2 in signal transduction of platelet derived growth factor in vascular smooth muscle cells. Biochem J. 1993;290:649-653.
4.
Morrero MB, Paxton WG, Duff JL, Berk BC, Berstein KE. Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-1 in vascular smooth muscle cells. J Biol Chem. 1994;269:10935-10939.
5.
Resink TJ, Scott-Burden T, Baur U, Burgin M, Buhler FR. Enhanced responsiveness to angiotensin II in vascular smooth muscle cells from spontaneously hypertensive rats is not associated with alterations in protein kinase C. Hypertension. 1989;14:293-303.
6. Paquet JL, Banomin-Legros M, Brunelle G, Meyer P. Angiotensin II induced proliferation of aortic myocytes in spontaneously hypertensive rats. J Hypertens. 1990;8:565-572.[Medline] [Order article via Infotrieve]
7.
Osani J, Dunn MJ. Phospholipase C responses in cells from spontaneously hypertensive rats. Hypertension. 1992;19:446-455.
8.
Bunkenberg B, Amelsvoort T, Rogg H, Wood JM. Receptor mediated effects of angiotensin II on growth of vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension. 1992;20:746-754.
9. Morton C, Baines R, Masood I, Ng L, Boarder MR. Stimulation of two vascular smooth muscle derived cell lines by angiotensin II: differential second messenger responses leading to mitogenesis. Br J Pharmacol. 1995;115:361-367.[Medline] [Order article via Infotrieve]
10. Bolger CT, Liard F, Jodoin A, Jamarillo J. Vascular reactivity, tissue levels and binding sites for endothelin: a comparison in the spontaneously hypertensive and Wistar-Kyoto rats. Can J Physiol Pharmacol. 1990;69:406-413.
11. Rapaport R. Cyclic guanosine monophosphate inhibition of contraction may be mediated through inhibition of phosphoinositide hydrolysis in rat aorta. Circ Res. 1986;18:407-410.
12.
Lang D, Lewis MJ. Endothelium-derived relaxing factor inhibits the formation of inositol trisphosphate by rabbit aorta. J Physiol (Lond). 1989;411:45-52.
13.
Hirata M, Kohse KP, Chang C, Tkebe T, Murad F. Mechanisms of cyclic GMP inhibition of inositol phosphate formation in rat aorta segments and cultured bovine aortic smooth muscle cells. J Biol Chem. 1990;265:1268-1273.
14. Watt PAC, Thurston H. Endothelium-dependent relaxation in resistance vessels from the spontaneously hypertensive rat. J Hypertens. 1989;7:661-666.[Medline] [Order article via Infotrieve]
15. Fukada N, Izumi Y, Minato M, Soma M, Watanabe Y, Watanabe M, Itatano M. Cyclic GMP formation of resistance vessels in the development of hypertension. Jpn Circ J. 1991;55:721-728.[Medline] [Order article via Infotrieve]
16. Legrand MCM, Benessiano J, Levy BI. Endothelium, mechanical compliance and cyclic GMP content in the carotid artery from spontaneously hypertensive rats. J Cardiovasc Pharmacol. 1993;21(suppl 1):526-530.
17.
Koller A, Huang A. Impaired nitric oxide-mediated flow-induced dilation in arterioles of spontaneously hypertensive rats. Circ Res. 1994;74:416-421.
18.
Tesfamariam B, Halpern W. Endothelium-dependent and endothelium-independent vasodilatation in resistance arteries from hypertensive rats. Hypertension. 1988;11:440-444.
19. Kahonen M, Makynen H, Wu X, Arvola P, Porsti I. Endothelial function in spontaneously hypertensive rats: influence of quinapril treatment. Br J Pharmacol. 1995;115:859-867.[Medline] [Order article via Infotrieve]
20. Davies JE, Ng LL, Ameen M, Syme PD, Aronson JK. Evidence for altered Na+/H+ antiport activity in cultured skeletal muscle cells and vascular smooth muscle cells from spontaneously hypertensive rats. Clin Sci. 1991;80:509-518.[Medline] [Order article via Infotrieve]
21. Challiss RAJ, Batty I, Nahorski SR. Mass measurements of inositol (1,4,5)trisphosphate in rat cerebral cortex slices using a radioreceptor assay: effects of neurotransmitters and depolarisation. Biochem Biophys Res Commun. 1988;157:684-691.[Medline] [Order article via Infotrieve]
22.
Grynkiewicz G, Poenie M, Tsien RY. A new generation of calcium indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-3450.
23. Brooker G, Harper JF, Terasaki WL, Moylan RD. Radioimmunoassay for cyclic GMP and cyclic AMP. Adv Cyclic Nucleotide Res. 1979;10:1-33.[Medline] [Order article via Infotrieve]
24.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
25.
Fujii K, Tominaga M, Ohmori S, Kobayashi K, Koga T, Takata Y, Fujishima M. Decreased endothelium-dependent hyperpolarization to acetylcholine in smooth muscle of the mesenteric artery of spontaneously hypertensive rats. Circ Res. 1992;70:660-669.
26.
Luscher TM, Vanhoutte PM. Endothelium dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension. 1986;8:344-348.
27. Jameson M, Dai F-X, Luscher T, Skopec J, Dietrich A, Dietrich D. Endothelium derived contracting factors in resistance arteries of young spontaneously hypertensive rats before development of overt hypertension. Hypertension. 1993;21:326-332.
28. Resink TJ, Scott-Burden T, Baur U, Buhler F. Atrial natriuretic peptide: binding and cyclic AMP response in cultured vascular smooth muscle cells from spontaneously hypertensive rats. Am J Hypertens. 1989;2:32-39.[Medline] [Order article via Infotrieve]
29.
Bian K, Bukoski D. Myofilament calcium sensitivity of normotensive and hypertensive resistance arteries. Hypertension. 1995;25:110-116.
30. Pasanisi F, Hamilton CA, Wadsworth R, Pryce C, Nixon G, Reid JL. Phosphoinositide metabolism and ageing in aorta from SHR and WKY rats: effects of endothelin-1 and noradrenaline. Clin Exp Hypertens. 1994;16:119-133.
31. Thorin-Trescases N, Hamilton CA, Jardine E, Reid JL. Signal transduction mechanisms of the vasoconstriction in hypertension. Eur J Pharmacol. 1994;268:199-207.[Medline] [Order article via Infotrieve]
32. Sugiyama T, Yoshizumi M, Takafu F, Urabe H, Tsukakoshi M, Kasuya T, Yasaki Y. The elevation of the cytoplasmic calcium ions in vascular smooth muscle cells in SHR: measurement of the free calcium ions in single living cells by laser microspectrofluorimetry. Biochem Biophys Res Commun. 1986;141:340-346.[Medline] [Order article via Infotrieve]
33.
Turla MB, Webb RC. Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats. Am J Physiol. 1990;258:H173-H178.
34. Vila E, Macrae IM, Reid JL. Differences in inositol phosphate production in blood vessels of normotensive and spontaneously hypertensive rats. Br J Pharmacol. 1991;104:296-300.[Medline] [Order article via Infotrieve]
35. Guild SB, Jenkinson S, Muir TC. Noradrenaline-stimulated inositol phosphate accumulation in arteries from spontaneously hypertensive rats. Br J Pharmacol. 1992;106:859-865.[Medline] [Order article via Infotrieve]
36.
Gopalakrishna R, Chen ZH, Gundimeda U. Nitric oxide generating agents induce reversible inactivation of protein kinase C activity and phorbol ester binding. J Biol Chem. 1993;268:27180-27185.
37. Parker PJ, Hemmings BA, Gierschik P. PH domains and phospholipases: a meaningful relationship? Trends Biochem Sci. 1994;19:54-55.[Medline] [Order article via Infotrieve]
38.
Banno Y, Okano Y, Nozawa Y. Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-1. J Biol Chem. 1994;269:15846-15852.
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