Norepinephrine-induced calcium signaling pathways in afferent arterioles of genetically hypertensive rats

Max Salomonsson and William J. Arendshorst

Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545


    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
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This study provides new information about the relative importance of calcium mobilization and entry in the renal vascular response to adrenoceptor activation in afferent arterioles isolated from 7- to 8-wk-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Intracellular free calcium concentration ([Ca2+]i) was measured in microdissected arterioles utilizing ratiometric photometry of fura 2 fluorescence. There was no significant strain difference in baseline [Ca2+]i. Norepinephrine (NE; 10-6 and 10-7 M) elicited immediate, sustained increases in [Ca2+]i. The general temporal pattern of response to 10-6 M NE consisted of an initial peak and a maintained plateau phase. The response to NE was partially blocked by nifedipine (10-6 M) or 8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate (TMB-8; 10-5 M). A calcium-free external solution abolished the sustained [Ca2+]i plateau response to NE, with less influence on the peak response. In the absence of calcium entry, TMB-8 (10-5 M) completely blocked the calcium response to NE in WKY but not SHR, suggesting strain differences in mobilization. A higher concentration of TMB-8 (10-4 M), however, blocked all discernible mobilization in both strains. We conclude that there are differences in Ca2+ handling in renal resistance vessels between young WKY and SHR with respect to mobilization stimulated by alpha -adrenoceptors. Afferent arterioles of young SHR appear to have a larger inositol-1,4,5-trisphosphate-sensitive pool or release from a site less accessible to TMB-8.

spontaneously hypertensive rats; Wistar-Kyoto rats; norepinephrine; hypertension; 8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate; nifedipine; vascular smooth muscle; inositol-1,4,5-trisphosphate-mediated mobilization; L-type calcium channel; renal circulation


    INTRODUCTION
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INTRODUCTION
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IT IS WELL KNOWN THAT HYPERTENSION is associated with increased peripheral vascular resistance. Less clear, however, is whether the increased vasomotor tone is a primary abnormality or whether it develops secondarily to the increased pressure. Cross-transplantation of kidneys between hypertensive and normotensive rat strains leads to development of hypertension in previously normotensive animals and to normalization of the elevated blood pressure in the hypertensive-prone strain. This indicates a crucial role of the kidneys and has been shown for various models of hypertension including very young spontaneously hypertensive rats (SHR) (39). The precise mechanism responsible for the renal defect is elusive, but the kidneys are known to be vasoconstricted during development of hypertension at the age of 6-8 wk in association with reduced glomerular filtration rate (GFR) and retention of salt and water (13, 20, 37). Furthermore, kidneys of young SHR exhibit increased reactivity to vasoconstrictors such as angiotensin II (ANG II), arginine vasopressin (AVP), and thromboxane A2 (8, 15). Increased extracellular fluid volume and autoregulatory- and paracrine-induced vasoconstriction in the peripheral circulation are likely to contribute to, if not cause, the elevated arterial pressure (24).

The renal circulation is richly endowed with sympathetic nerves, and the autonomic nervous system is poised to play an important role in the control of renal hemodynamics, GFR, extracellular fluid volume, and arterial blood pressure (3, 11). The critical function of renal nerves is indicated during the onset of hypertension in SHR. Renal denervation delays or attenuates the development of hypertension in genetic models (29) in association with exaggerated denervation natriuresis (40). With regard to renal vascular reactivity to adrenergic stimulation in genetic hypertension, studies to date have been less than conclusive. Some of the variation in results may result from the wide range of experimental conditions and age of the animals (4, 9, 12, 17, 45, 50).

Catecholamines released from nerve terminals and of hormonal origin exert their effects by activation of cell surface adrenoceptors on vascular smooth muscle cells (VSMC) to produce changes in cytosolic calcium concentration ([Ca2+]i) and the resultant contractile response. Activation of alpha -adrenoceptors induces an increase in [Ca2+]i by recruitment of calcium from one or two major sources, i.e., mobilization from intracellular stores, and/or entry from the extracellular space, in afferent and efferent arterioles (26, 42). Calcium entry may occur through voltage-dependent and/or receptor-activated calcium channels located in the plasma membrane; in some cell types, store-operated calcium entry depends on depletion of intracellular stores. VSMC exhibit two sites of internal calcium stores, the sarcoplasmatic reticulum and mitochondria (33), although the latter is thought to contribute little to the regulation of [Ca2+]i in VSMC. Two channels for release of calcium from sarcoplasmic reticulum are a ryanodine receptor and an inositol-1,4,5-trisphosphate (IP3) receptor. The IP3 receptor appears to be present in all VSMC, whereas the contribution of the ryanodine receptor is poorly understood. Evidence indicates that the relative contributions of entry vs. mobilization in the response to stimulation with cathecholamines differ in VSMC depending on location and function (7, 36).

Reports from work in several animals (e.g., rat, dog, cat, and monkey) have shown that an increase in renal sympathethic nerve activity decreases renal blood flow (RBF) and increases renal vascular resistance (11). Calcium entry blockers are known to block, at least in part, NE-induced renal vasoconstriction (25). Several studies have reported effects of norephinephrine (NE) on [Ca2+]i in isolated renal vessels (30, 42, 43). In this regard, we have previously shown in Sprague-Dawley rats that both the NE-induced [Ca2+]i response in isolated afferent arterioles and the renal vasoconstriction in vivo are attenuated ~50% by nifedipine (42). Moreover, the putative blocker of IP3-mediated calcium mobilization 8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate (TMB-8) inhibited NE-induced renal vasoconstriction or changes in [Ca2+]i by ~80% in both in vivo and in vitro preparations.

Differences in VSMC calcium handling may contribute to the increased vascular tone found in animals with genetic hypertension, during both developmental and established phases of hypertension. Evidence to date implicates strain differences in basal levels of [Ca2+]i (49), function of intracellular calcium stores (10, 35), and activity of calcium and potassium channels (41, 51). It should be recognized that most of these reports pertain to cultured VSMC obtained from large conduit vessels of relatively old SHR with established hypertension.

The present study was designed to identify mechanisms responsible for mobilization and entry of [Ca2+]i in response to stimulation of alpha -adrenoceptors by NE in renal resistance vessels microdissected from young normo- and hypertensive rats. The contribution of calcium entry pathways was tested using a nominally calcium-free bathing solution. The involvement of entry through voltage-sensitive L-type calcium channels was assessed using the pharmacological agent nifedipine. TMB-8 was used to inhibit IP3-dependent mobilization of calcium from internal stores. [Ca2+]i was measured in vitro in isolated afferent arterioles from 7- to 8-wk old SHR and WKY rats using the ratiometric fluorescence of the indicator fura 2.


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Glomeruli with an attached isolated afferent arteriole were microdissected from 7- to 8-wk-old WKY and SHR rats (weighing 208 ± 7 and 209 ± 5 g, respectively) from the Chapel Hill or Harlan colonies using methods described previously (42). Briefly, thin slices (thickness 0.5-1 mm) were cut from the midregion of the kidney. They were transferred to a dissection dish containing a chilled physiological salt solution (PSS) solution with BSA (Sigma) at a final concentration of 0.5 g/dl. The PSS solution had the following composition (mM): 135 NaCl, 5.0 KCl, 1.0 CaCl2, 1.0 MgCl2, 10 HEPES, and 5.0 D-glucose. Forceps were used for the isolation procedure under microscopic visualization (magnification ×12-100). The interlobular artery was localized at its origin from an arcuate artery, and a segment consisting of glomeruli, blood vessels, and tubular structures was removed. The tubular structures were carefully stripped away using forceps. A single afferent arteriole was cut at the bifurcation from an interlobular artery by using a knife blade. To obtain a homogenous population of superficial arterioles, we dissected arterioles from the outer one-third of the cortex. If no satisfactory preparation was obtained during the first 120 min of dissection, the kidney was discarded.

After the dissection procedure was completed, an afferent arteriole was loaded with fura 2-acetoxymethyl ester (AM) for 45-60 min at room temperature in the dark, as previously described (42). Fura 2-AM (Molecular Probes) was prepared as a stock solution in DMSO (1 mM) and mixed with PSS to give a final concentration of 2 µM and Pluronic F127 (Molecular Probes) (0.01%) immediately before use. After the loading was completed, the vessel was transferred to a chamber containing PSS on the stage of an inverted microscope (Olympus IX 70) using an Eppendorf micropipette. The proximal end of the arteriole and the glomerulus were then aspirated into concentric glass holding pipettes to maintain mechanical stability. For measurements of [Ca2+]i, the arteriole was centered in the optical field of ×40 quartz oil immersion objective, and shutters were adjusted to get an arteriole in a sampling window. The preparation was continuously visualized by the use of a video camera (Sony) and monitor. A dual-excitation wavelength DeltaScan equipped with dual monochromators and a light pathway chopper (Photon Technology International, NJ) was used for exciting with ultraviolet light of, alternatively, 340- and 380-nm wavelength. The fluorescent emission was detected by a photometer and was processed and stored by an IBM-compatible Pentium computer and Felix software (Photon Technology International). [Ca2+]i was calculated using the Grynkiewicz equation (22): [Ca2+]i = Kd · [(R-Rmin)/(Rmax-R)] · (Sf/Sb), where Kd is the dissociation constant of fura 2 for calcium; Sf and Sb are 380-nm fluorescence at zero and saturating calcium concentrations, respectively; and Rmin and Rmax are values of R (fluorescence ratio 340/380) at low and at saturating calcium concentration, respectively. Values for Kd, Rmin, Rmax, Sf, and Sb were determined in vitro as previously described (27, 42).

During replacement of fluids, the volume in the experimental chamber was maintained constant by the use of a vacuum suction system. All experiments were performed at room temperature (26-28°C). The experimental solutions were added by a volume large enough to allow total exchange of the composition in the experimental chamber several times. The nominally calcium-free solution was obtained by adding 1 mM EGTA to PSS and replacing CaCl2 with NaCl. Pilot studies established that 10-6 M of NE elicited one-half of maximal response in [Ca2+]i; this concentration is used throughout the present study unless stated otherwise. Nifedipine was dissolved in DMSO and diluted in PSS to a final concentration of 10-6 M, a concentration known to inhibit L-type calcium channels (18). TMB-8, an inhibitor of IP3-mediated calcium release from sarcoplasmic reticulum, was added to the same solutions to yield a final concentration of 10-5 or 10-4 M. The experiments were conducted as follows. Initially, the viability of each vascular preparation was determined by adding a short pulse (50 s) of NE. If there was no immediate [Ca2+]i response, the preparation was discarded. Inhibitory agents were added before or after NE stimulation. Arterioles were exposed to nifedipine (50 s), calcium-free medium (50 s), or TMB-8 (90 s) or combinations thereof before and during NE stimulation. The mean prestimulation [Ca2+]i values during the control period were obtained between 10 and 15 s before the addition of NE. Similarly, [Ca2+]i was measured as the immediate peak response (maximal peak value during the first 15 s after the addition of NE) and between 30 and 35 s (sustained plateau phase) after the stimulation was initiated. To establish reversibility and to exclude possible prolonged action and crossover effects of a particular pretreatment, the experiments were performed in random order.

Statistical analyses. Data are presented as means ± SE. The SigmaStat (Jandel Scientific/SPSS) software was used for data analysis. Statistical significance was evaluated by ANOVA for repeated measurements and the post hoc Newman-Keuls test. Student's unpaired t-test was used for strain difference. A P value of < 0.05 is considered statistically significant.


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Measurements of basal afferent arteriolar [Ca2+]i and the response to NE. Resting or prestimulation values of [Ca2+]i did not differ between afferent arterioles from 7- to 8-wk-old WKY (n = 22 arterioles from 18 rats) and age-matched SHR (n = 21 arterioles from 19 rats): 97 ± 6 vs. 81 ± 10 nM (P > 0.1). Stimulation with NE (10-6 M), which elicits a half-maximal response, caused an abrupt, step increase in [Ca2+]i in afferent arteriolar VSMC of WKY and SHR (Fig. 1). The general response is characterized by a sharp rise with a transient peak followed by a sustained plateau phase that remains 50-100 nM above baseline. Overall, the basic response pattern was similar in preparations from WKY and SHR. To more systematically analyze the responses, we selected the time points of the initial peak [Ca2+]i at 0-15 s and the average value recorded during the sustained plateau phase between 30 and 35 s. Preliminary studies established that the response at 30 s was commonly maintained for several minutes. Contraction of the arteriole observed on the video monitor correlated temporally with the increase in [Ca2+]i.


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Fig. 1.   Original recording of cytosolic calcium concentration ([Ca2+]i) in afferent arterioles depicting the stimulatory effect of norepinephrine (NE) (10-6 M) and the antagonism afforded by pretreatment with a combination of 8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate (TMB-8) (10-5 M) and a calcium-free EGTA (1 mM)-containing solution in Wistar-Kyoto (WKY) (A) and spontaneously hypertensive rats (SHR) (B). Note that the NE response is totally blocked after the pretreatment period in WKY but not in SHR.

In afferent arterioles from WKY, NE (10-6 M) caused a peak transient in [Ca2+]i that reached ~108% (from 103 ± 9 to 215 ± 16 nM) above baseline and a sustained plateau of ~67% (173 ± 12 nM, n = 22). The response in SHR arterioles was similar, amounting to an ~109% (from 85 ± 7 to 178 ± 15 nM) and ~61% increase (137 ± 9 nM) above resting levels, respectively (n = 21). The values for the immediate peak were always greater than those for the sustained plateau (P < 0.001); this was true for both strains. To investigate the possible strain difference in sensitivity to NE, we stimulated vessels with a lower concentration of NE (10-7 M). The lower concentration produced substantially smaller changes in [Ca2+]i than those elicited by 10-6 M NE. In WKY, [Ca2+]i rose from 94 ± 9 to an initial peak of 110 ± 11 nM, followed by a plateau level of 106 ± 12 nM (n = 11). In SHR, NE increased [Ca2+]i from 70 ± 7 to immediate peak values of 90 ± 9 nM and then a sustained plateau of 82 ± 7 nM (n = 10). All changes compared with baseline were statistically significant in WKY and SHR (P < 0.01). However, only in arterioles from SHR was the absolute difference between peak and plateau levels statistically significant (P < 0.05).

NE-induced calcium entry. To evaluate the importance of extracellular calcium in the NE response, the effects of NE were assessed with and without a calcium-free bath solution containing EGTA (1 mM). WKY arterioles in the normal calcium-containing solution responded to NE (10-6 M) with increases in [Ca2+]i from 134 ± 27 to a peak value of 237 ± 38 nM (~78% increase above baseline) between 0 and 15 s and to 200 ± 31 nM (~50% increase above baseline) at 30-35 s (n = 5, P < 0.001). After short-term (60 s) exposure to the calcium-free solution, the small decrease in baseline [Ca2+]i (from 128 ± 25 to 106 ± 27 nM) was not statistically significant (P = 0.16). NE challenge in the calcium-free solution caused a transient peak in [Ca2+]i to ~54% above baseline (to 163 ± 36 nM, P < 0.01, n = 5). The calcium values during continued NE stimulation for 30-35 s declined to control levels (110 ± 25 vs. 106 ± 27 nM) (Fig. 2). Thus NE stimulation was significantly attenuated when calcium entry was limited by calcium-free extracellular fluid, especially during the sustained plateau phase (P < 0.001). SHR afferent arterioles in the presence of normal extracellular calcium responded to NE (10-6 M) with increases in [Ca2+]i from 107 ± 18 to a peak value of 208 ± 27 nM (~95% increase above baseline) and to a sustained plateau level of 168 ± 20 nM at 30-35 s (~57% increase, n = 5, P < 0.001). After pretreatment of SHR vessels with Ca2+-free solution for 60 s, resting [Ca2+]i was relatively stable, falling slightly (from 96 ± 21 to 72 ± 15 nM) (P = 0.6), and NE elicited an increase of ~138% (to 171 ± 45 nM). At 30-35 s, [Ca2+]i had declined to prestimulation levels (80 ± 14 nM). Thus in contrast to WKY, the mean absolute peak response of roughly 100 nM in SHR was not significantly attenuated by pretreatment with a calcium-free solution.


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Fig. 2.   The peak and sustained plateau (30 s) changes in [Ca2+]i after stimulation with NE (10-6 M) before and after pretreatment with a nominally calcium-free solution containing EGTA (1 mM). Experiments are performed in afferent arterioles from WKY and SHR. The values are NE-induced absolute increases in [Ca2+]i from baseline in the presence or absence of the pretreatment calcium-free solution. Values are means ± SE. Each group consists of 5 arterioles. *P < 0.05 vs. baseline; #P < 0.05 vs. NE without treatment.

Calcium release from intracellular stores. To further assess the involvement of calcium mobilization, the response to NE was assessed before and during treatment with TMB-8, an inhibitor of IP3-induced calcium release (38). Before drug application, NE elicited a peak increase in [Ca2+]i (108 ± 13 to 256 ± 28 nM) at 0-15 s and a sustained response at 210 ± 22 nM at 30-35 s (P < 0.001) in 7 WKY preparations. Ninety seconds of exposure to TMB-8 did not affect baseline [Ca2+]i but did attenuate NE effects on [Ca2+]i. TMB-8 inhibited both the peak and plateau responses (P < 0.01). As Fig. 3 shows, [Ca2+]i rose from 101 ± 12 to 178 ± 24 nM between 0 and 15 s and to 135 ± 13 nM at 30-35 s (P < 0.05, n = 7). Likewise, the NE-induced response was attenuated by TMB-8 in afferent arterioles of SHR; baseline values were unaffected. In SHR control experiments, NE caused an immediate rise in [Ca2+]i from 102 ± 12 to 221 ± 28 nM, with a sustained elevation (167 ± 15 nM at 30-35 s, P < 0.001, n = 8). After TMB-8 pretreatment, NE produced an initial rise of 82% above baseline (90 ± 12 to 164 ± 20 nM) that remained elevated above control (113 ± 12 nM) at 30-35 s (P < 0.05, n = 8).


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Fig. 3.   Summarized data showing the effects of a blockade of inositol 1,4,5-trisphosphate-mediated intracellular calcium release with TMB-8 (10-5 M) on peak and sustained plateau [Ca2+]i after stimulation with NE (10-6 M). Data are from afferent arterioles of WKY and SHR. The values are NE-induced increases from baseline [Ca2+]i. Values are means ± SE. Each group consists of 7 or more arterioles. *P < 0.05 vs. baseline; #P < 0.05 vs. NE without treatment.

TMB-8 sensitivity of calcium release. To examine sensitivity of calcium release to TMB-8, afferent arterioles were pretreated for 90 s with a combination of TMB-8 (10-5 M or 10-4 M) and a calcium-free solution + 1 mM EGTA. Representative tracings are shown in Fig. 1. In control experiments in WKY arterioles (n = 7), NE caused an initial increase from 102 ± 14 to 236 ± 26 nM, followed by a sustained plateau of 189 ± 16 nM at 30-35 s (P < 0.01). After pretreatment with the lower concentration of TMB-8 (10-5 M) in the calcium-free solution, the response to NE was completely abolished (Fig. 1 A; Fig. 4). Basal [Ca2+]i declined to 72 ± 10 nM on average, and no significant changes were noted during NE stimulation at 0-15 s (89 ± 15 nM) and at 30 to 35 s (76 ± 11 nM, P > 0.2). In contrast to the complete inhibition in WKY, responses were clearly evident in SHR arterioles. Here we found that NE still elicited an immediate peak response in the presence of TMB-8 and the calcium-free solution. In the SHR control experiments, NE caused a peak increase in [Ca2+]i of 169 nM above baseline (100 ± 14 to 269 ± 28 nM) and a sustained plateau level (179 ± 22 nM, n = 6, P < 0.01). Combined TMB-8 treatment with the calcium-free solution for 90 s did not significantly reduce baseline [Ca2+]i (from 97 ± 14 to 63 ± 12 nM, P = 0.07). Subsequent stimulation with NE caused a significant peak increase of 59 nM (to 122 ± 22 nM, P < 0.001) with a subsequent return to baseline at 30 s (69 ± 12 nM). A higher concentration of TMB-8 (10-4 M) totally abolished the intracellular release in both strains. In WKY, basal [Ca2+]i in the presence of TMB-8 and the EGTA-containing, calcium-free solution was 82 ± 10 nM (n = 4). Subsequent stimulation with NE did not affect [Ca2+]i, which remained at 80 ± 8 nM. In SHR, the corresponding values were 60 ± 7 and 60 ± 8 nM (n = 7). Thus the results from this series of experiments suggest that afferent arterioles of young SHR exhibit a lower sensitivity to TMB-8 (Fig. 5).


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Fig. 4.   The peak and sustained plateau changes in [Ca2+]i after stimulation with NE (10-6 M) before and after pretreatment with a combination of TMB-8 (10-5 M) and a calcium-free, EGTA-containing (1 mM) solution in afferent arterioles from WKY and SHR. Values are NE-induced absolute increases in [Ca2+]i in the presence or absence of the pretreatment solution. Note that there is a statistically significant NE effect after pretreatment in SHR only. Values are means ± SE. Each group consisted of 6 or more arterioles. *P < 0.05 vs. baseline; #P < 0.05 vs. NE without treatment; $P < 0.05 vs. the same treatment in WKY.



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Fig. 5.   Group averages showing the transient [Ca2+]i peak response to NE in the presence of calcium-free solution and TMB-8 (10-5 or 10-4 M). Note that the low-TMB-8 concentration completely blocks in WKY whereas the higher concentration is required in SHR. *P < 0.001 vs. baseline.

Involvement of voltage-operated calcium channels. To investigate the involvement of voltage-gated L-type calcium channels the dihydropyridine nifedipine was used to antagonize the NE-induced [Ca2+]i response. In WKY before nifedipine treatment, NE caused the usual peak and plateau responses of 145 and 87 nM (85 ± 10 to 230 ± 24 nM and then 172 ± 26 nM; P < 0.01; n = 6) (Fig. 6). Pretreatment with nifedipine (10-6 M) for 60 s did not influence basal [Ca2+]i (P > 0.4); however, it attenuated both the peak and plateau response to NE by ~50% (P < 0.05). As Fig. 6 shows, for WKY vessels treated with the calcium channel blocker, the peak [Ca2+]i response to NE was attenuated to 80 nM and the pleateau phase to 33 nM; the absolute [Ca2+]i values rose from 72 ± 12 to 152 ± 11 nM and then declined to 105 ± 6 nM (P < 0.05 vs. nifedipine). In afferent arterioles from SHR (n = 6), NE in the control period increased [Ca2+]i from 71 ± 7 nM to an initial peak (204 ± 23 nM) and a plateau (144 ± 9 nM) that were 187 and 102% above baseline, respectively (P < 0.01). In the experimental period, nifedipine pretreatment did not significantly decrease basal [Ca2+]i (68 ± 7 to 57 ± 5 nM, P > 0.05); however, it attenuated the responses to NE, reducing the immediate peak response to 152% above baseline (144 ± 24 nM) and the sustained plateau to 53% above control (87 ± 6 nM) (P < 0.05). Thus nifedipine attenuated the NE-induced [Ca2+]i response to the same extent in WKY and SHR.


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Fig. 6.   Effect on peak and sustained plateau in [Ca2+]i of stimulation with NE (10-6 M) in the presence and absence of nifedipine (10-6 M) in afferent arterioles from WKY and SHR. The values are NE-induced absolute increases in [Ca2+]i from baseline in the presence or absence of the nifedipine-containing solution. Values are means ± SE. Each group consisted of 6 arterioles. *P < 0.05 vs. baseline; #P < 0.05 vs. NE without treatment.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study presents new information about the cellular mechanisms mediating adrenoceptor-induced activation of cytosolic calcium in VSMC of resistance vessels in the renal microcirculation. One objective was to determine the relative importance of entry of extracellular calcium and mobilization of calcium from intracellular stores in microdissected afferent arterioles. A related aim was to investigate possible strain differences in these calcium signaling pathways between young WKY and SHR. The present studies extend to afferent arterioles of young WKY and SHR our previous findings on calcium signaling in the control of renal vascular resistance in vivo and cytosolic calcium in afferent arterioles of Sprague-Dawley rats (42). Our earlier work established that the vasoconstrictor actions of NE involve a combination of calcium entry that is antagonized by conventional calcium channel blockers with calcium mobilization sensitive to an antagonist of the IP3-mediated pathway. NE-induced renal vasoconstriction was attenuated in a dose-dependent manner by dihydropyridine nifedipine, with maximal ~50% inhibition of both the NE-induced renal vasoconstriction and the increase in arteriolar [Ca2+]i. TMB-8 blocked ~80% of the NE response in vivo and in vitro. In vivo blood flow experiments showed that there was no additive effect of combined treatment with nifedipine and TMB-8, indicating a continuous and complex interplay between calcium mobilization and entry.

In the present study, the [Ca2+]i response to NE (10-6 M) was similar in afferent arterioles isolated from 7- to 8-wk-old WKY and SHR. However, the overall response pattern, characterized by an initial peak followed by a sustained plateau phase, differed somewhat from what we previously found in Sprague-Dawley rats (42). In our earlier work, the majority of the [Ca2+]i responses to NE were square shaped with the initial peak and sustained plateau of similar magnitudes. Both WKY and SHR exhibited a slightly more pronounced initial transient peak relative to the maintained plateau phase. It is not clear whether the response patterns reflect strain differences or the fact that younger animals were used in the present study. The methods utilized for microdissection of afferent arterioles and the measurement of fura 2 fluorescence were identical.

It is noteworthy that a lower concentration of NE (10-7 M) also elicited equivalent [Ca2+]i changes in arterioles from WKY and SHR. In both strains there was a small but clearly significant increase in response to this concentration of NE. However, only in SHR was there a statistically significant difference between the peak and plateau recordings of [Ca2+]i using this lower dose of NE. We cannot, however, convincingly conclude that this reveals a more pronounced susceptibility of the SHR to stimulation with NE in the form of an initial transient [Ca2+]i response.

There are conflicting reports in the literature regarding the magnitude of the constrictor response to adrenergic stimulation of VSMC from WKY and SHR. For example, in one study utilizing the isolated perfused kidney preparation, administration of NE or nerve stimulation caused more pronounced renal vasoconstriction in young (4-5 wk) SHR than in WKY (45). However, the contractile sensitivity to NE was reduced in SHR. In other isolated kidney studies, increased reactivity to NE was found in both young (4 wk) and adult (2 and 4 mo) stroke-prone SHR compared with age- and sex-matched WKY (4). On the other hand, reduced renal vascular reactivity of ex vivo blood-perfused kidneys to NE has been reported for 12- to 16-wk-old SHR compared with WKY (17), whereas other investigators found no strain difference for this age group in vivo (12).

It has been proposed that VSMC from hypertensive rats have abnormalities in calcium metabolism (44). Dysfunction may involve intracellular overload of calcium (19), handling of calcium by sarcoplasmic reticulum (5, 10, 31), ion channels (32), or an Na+/Ca2+ exchanger (34). In the present study, we used young rats at 7 to 8 wk of age to characterize SHR during the developmental phase of hypertension, characterized by rapid increases in arterial pressure and relative retention of salt and water (13). In contrast to what other investigators have found in VSMC from other vascular beds (47), baseline [Ca2+]i did not differ statistically from normal in SHR afferent arterioles, although it tended to be slightly lower than WKY values. Our observations agree with the lack of a strain difference in baseline [Ca2+]i found in small muscular arteries (15-35 µm outer diameter) (46) and cultured aortic VSMC (35). Furthermore, no differences in basal [Ca2+]i levels were found between cultured VSMC from mesenteric resistance arteries during primary and first passages (6). Interestingly, basal [Ca2+]i rose in VSMC from SHR during subculture to beyond passage 3 (6). Clearly, reports of possible strain differences in basal or stimulated [Ca2+]i need to be qualified by detailed experimental conditions and interpreted with caution. Furthermore, because the morphology of the resistance vessels may differ between preparations from WKY and SHR, one cannot completely exclude structural modifications and their potential impact on optical properties and measurements of dye fluorescent emission (37).

We have previously shown in Sprague-Dawley rats that the NE-induced [Ca2+]i response in isolated afferent arterioles exhibited a similar ~50% attenuation by nifedipine (42). These results indicated the importance of L-type voltage-gated calcium channels in the rat renal vascular response to activation of adrenoceptors. The existence of voltage-gated calcium channels was further supported by the fact that potassium-induced depolarization of the isolated arterioles caused calcium entry exclusively via a nifedipine-sensitive pathway. However, we did not find any significant alterations of the baseline [Ca2+]i by administration of nifedipine in Sprague-Dawley rats.

Alterations of calcium channels may at least partially account for variability seen in baseline [Ca2+]i, and the [Ca2+]i response to agonist activation between hypertensive and normotensive animals. Support for that notion has been provided by several laboratories (32, 48). In the present study on vessels from young rats, pretreatment with nifedipine attenuated the response to NE to the same extent in WKY and SHR. Nifedipine attenuated the sustained plateau response to NE by roughly 50% when extracellular calcium concentration was normal, as previously demonstrated for renal arterioles of Sprague-Dawley rats (42). Thus our findings indicate that dihydropyridine-sensitive calcium channels contribute to the NE-induced [Ca2+]i response to the same extent in SHR and WKY. The fact that low extracellular calcium inhibited the NE-induced sustained increase in [Ca2+]i more strongly than nifedipine implicates a second calcium entry pathway, present in SHR and WKY, as previously found in renal arterioles of Sprague-Dawley rats (42). By definition, calcium entry via this pathway is insensitive to the dihydropyridine class of agents and thus independent of L-type calcium channels. Previous studies provide support for the participation of more than one calcium entry in smooth muscle preparations of different vascular origin, e.g., store-operated capacitative entry (14, 21). Such mechanisms may be responsible for distinction between the effects of nifedipine and the calcium-free medium. It is possible that exposure to a calcium-free medium could influence calcium release from internal stores. Our preliminary studies reveal, however, that a [Ca2+]i response to NE exists after several min of pretreatment with a calcium-free solution.

With regard to the relative importance of calcium mobilization from intracellular stores, we tested TMB-8, an inhibitor of IP3-mediated calcium release from intracellular stores, on the NE-induced [Ca2+]i response (38). We found that pretreatment with TMB-8 attenuated both the peak and the plateau increases in [Ca2+]i produced by NE. Both were inhibited to the same extent in WKY and SHR, in accord with previous findings for Sprague-Dawley rats (42). A generally accepted model for VSMC predicts that calcium entry is solely responsible for the sustained phase of [Ca2+]i increase and that inhibition of calcium mobilization, an initial event, thus should not interfere with the sustained stimulation. In our present studies, it is clear that the plateau phase is totally dependent on extracellular calcium because this phase is absent in a calcium-free environment. The fact that TMB-8 attenuates both the immediate peak and the sustained [Ca2+]i plateau in the presence of extracellular calcium indicates a continuous interaction between IP3-induced mobilization and entry. This finding holds for afferent arterioles from WKY, SHR, and Sprague-Dawley rats. The fact that the peak is substantially higher in the presence than in the absence of extracellular calcium, at least in WKY vessels, indicates that the initial peak depends on entry as well as intracellular release. Calcium entry via L-type channels during the peak and plateau is indicated by nifedipine inhibition of both phases of calcium responses. Also, several reports differ from the "noninteracting" time-dependent model and support the notion that calcium recruitment pathways may vary between large-diameter conduit vessels and small-diameter resistance vessels (7, 36). It is difficult to draw any conclusions from the shape of the response and as to whether it consists of a peak and a plateau or a square-shaped response. A square-shaped response may have an initial response that consists of the same components as in the "peak-plateau type"; it just does not surpass the level of the sustained plateau. The specificity of TMB-8 to block the IP3 pathway has been questioned (1). Earlier studies from our laboratory have shown that the lower concentration of TMB-8 (10-5 M) used in this study has no demonstrable effect on the calcium entry through voltage-sensitive calcium entry channels in isolated arterioles (42). Furthermore, it has no effect on renal vasoconstriction elicited by stimulation of L-type calcium entry channels with BAY K 8644 in vivo (16).

If one assumes that the mobilization of calcium from intracellular stores relies solely on release from IP3-sensitive pathways and that this release is totally blocked by the employed low dose of TMB-8, no agonist-induced [Ca2+]i response should be expected after pretreatment with a combination of TMB-8 and a calcium-free solution. In the present study, these assumptions hold true for afferent arterioles of WKY. After pretreatment with both concentrations of TMB-8 in a calcium-free solution, there was no NE-induced response in afferent arterioles from WKY. In contrast, in SHR there was a clear transient response to NE after treatment with the lower concentration of TMB-8. On the other hand, the higher concentration of TMB-8 in combination with the calcium-free solution totally abolished the response to NE. For both strains and both concentrations used, the combination of TMB-8 and the calcium-free medium totally abolished the sustained plateau phase in both strains.

Thus the present results strongly suggest a difference in calcium handling of the intracellular calcium stores between SHR vs. WKY afferent arteriolar VSMC at the age of 7 to 8 wk. This might reflect either different properties of the IP3-sensitive pool or the contribution of a non-IP3-sensitive pool. In this regard, a 1.6-fold higher IP3 receptor activity has been found in aortic microsomes of SHR compared with WKY (5). Other investigators found an increased responsiveness to IP3 in cardiac myocytes from SHR relative to WKY (28). In cultured aortic myocytes from 9- to 10-wk-old rats, it is reported that thapsigargin causes a larger calcium release that might indicate a larger intracellular calcium pool (10). This study also reported an enhanced angiotensin II-induced release of calcium in calcium-free conditions in SHR and that this augmented mobilization could be blocked by ryanodine. This might indicate a larger contribution of a ryanodine-sensitive pool in SHR. Furthermore, the presence of additional thapsigargin- and nonthapsigargin-sensitive intracellular calcium pools in SHR vessels (35) has been suggested. A third possibility is that IP3 is accumulated to a higher degree by NE stimulation in SHR. This has indeed been suggested for VSMC from SHR, presumably due to a decreased rate of dephosphorylation of IP3, rather than an increased production (23).

It is therefore evident that there are several levels in the chain of events leading to agonist-induced release of calcium from intracellullar stores that may differ between VSMC of SHR and WKY. From our results, it is not possible to determine whether the intracellular calcium pool consists of one or two compartments. One interpretation is that one single mechanism accounts for the total release and that this mechanism is simply more sensitive to attenuation by TMB-8 in WKY. This notion is supported by the fact that the higher concentration of TMB-8 (10-4 M) totally abolished the effect of NE under calcium-free conditions. Another explanation is that there are two distinct release mechanisms, one of which is more pronounced in afferent arteriolar VSMC of SHR than in those of WKY and less sensitive to TMB-8. The sensitivity to the higher concentration of TMB-8 could then be due to a nonspecific effect, as previously suggested (1). On the other hand, we found in preliminary experiments, both in WKY and SHR, that the transient increase in [Ca2+]i stimulated by response to caffeine, an agent commonly used to activate a ryanodine receptor channel in a calcium-free solution, is essentially unaffected by the highest concentration of TMB-8 employed in the present study, which supports the notion that TMB-8 is selective, without general inhibition of all mobilization pathways. Other investigators have found that the effect of caffeine is not affected by TMB-8 in single VSMC from rat tail artery (2). If our preliminary finding holds true, this indicates that caffeine-sensitive receptors are not involved in the relatively TMB-8-sensitive response to NE in SHR. From the results obtained in the present study we cannot, however, totally discriminate between a single mechanism that is less TMB-8 sensitive in SHR or two distinct intracellular calcium pools. We were not able to detect any strain differences when blocking the NE-induced response with TMB-8 in the presence of extracellular calcium. This could be explained by the possibility that the calcium release mechanism(s) might be suppressed by an elevation of [Ca2+]i. It is possible that during TMB-8 treatment alone the NE-induced calcium influx via dihydropyridine-sensitive channels and/or the alternative entry pathway might account for the attenuation of the mobilization pathway in SHR. Further studies are required to provide more in-depth information about conditions leading to the existence of and the functional relative importance of the less TMB-8-sensitive, or additional calcium mobilization pathway, in VSCM of resistance arterioles.

In summary, our study presents novel findings regarding calcium signaling mechanisms during stimulation of adrenergic receptors in renal resistance vessels from 7- to 8-wk-old WKY and SHR. In both strains, NE-induced increases in [Ca2+]i were dependent on both calcium entry via dihydropyridine-sensitive calcium channels and on TMB-8-sensitive intracellular mobilization. Exposure of isolated vessels to a calcium-free solution results in marked attenuation of the NE-induced sustained increase in [Ca2+]i in arterioles from both strains; the fact that inhibition was more marked than the nifedipine effect suggests the operation of calcium entry pathways in addition to L-type channels. The fact that, in SHR, a [Ca2+]i response was induced after pretreatment with a combination of calcium-free solution and TMB-8 (low dose) might indicate a larger intracellular calcium pool, a single release mechanism that is less TMB-8 sensitive in SHR or an alternative pathway for calcium mobilization distinct from IP3. The observed strain differences in calcium signaling may represent a mechanism responsible for enhanced renal vascular reactivity in young SHR during the development of genetic hypertension.


    ACKNOWLEDGEMENTS

These studies were supported by National Heart, Lung, and Blood Institute Grant HL-02334. M. Salomonsson's visit was sponsored in part by the Swedish Medical Research Council, The Royal Physiographic Society in Lund, Medical Faculty Lund University, Maggie Stephens Foundation, and the Berth von Kantzow Foundation.


    FOOTNOTES

Present address of M. Salomonsson: Dept. of Medical Physiology, Univ. of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark.

Address for reprint requests and other correspondence: W. J. Arendshorst, Dept. of Cell and Molecular Physiology, CB #7545, School of Medicine, Rm. 152, Medical Sciences Research Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: arends{at}med.unc.edu).

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

Received 20 June 2000; accepted in final form 20 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 281(2):F264-F272
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