INTRODUCTION

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IT IS WELL KNOWN THAT agonist-induced changes in intracellular cytosolic calcium concentration ([Ca²⁺]_i) in smooth muscle cells (SMC) are involved in regulation of cell contraction and control of vascular resistance. Studies on vascular SMC from relatively large-conduit vessels, such as aorta and mesenteric artery, indicate that neural-hormonal agents such as angiotensin II (ANG II) and arginine vasopressin (AVP) interact with cell surface receptors to produce changes in [Ca²⁺]_i (16, 34, 40, 41, 44). Although small-diameter arterioles play a major role in the regulation of vascular resistance and control of local blood flow, there are few definitive studies of calcium signaling and the importance of specific second messenger systems in SMC of resistance vessels. It is often tacitly assumed that the basic mechanisms and their relative importance are similar in large-diameter arteries and small-diameter arterioles. Nevertheless, conduit and resistance vessels exhibit functional differences, and their composite SMC are likely to possess different physiological properties and respond differently to physiological stimuli. There is evidence to support the view that calcium signaling differs between aorta and peripheral vessels (11, 26).

Previous studies of specific signaling mechanisms leading to changes in cytosolic calcium concentration in renal vessels are largely inferred from reports on cultured glomerular mesangial cells. These cells become contractile in culture, and signaling mechanisms are often taken as a model of mechanisms that may operate in renal resistance vessels. The relative contributions of second messenger systems in cultured mesangial cells do not differ substantially from those evident in cultured SMC derived from large systemic arteries (1, 2, 5, 7, 12, 33, 38). Stimulation of cultured mesangial cells with agonists such as ANG II, AVP, and endothelin cause an immediate increase of [Ca²⁺]_i that is short-lived and rapidly returns to baseline values (3, 23, 43). This change in [Ca²⁺]_i is primarily mediated by calcium mobilization, independent of calcium entry.

In the kidney, the afferent arteriole, a major site of resistance, is of primary importance in regulating arterial pressure and renal blood flow, as well as glomerular filtration rate. The available evidence suggests calcium signaling in renal resistance vessels may involve calcium mechanisms dependent on variable degrees of calcium entry from the extracellular compartment and calcium mobilization from internal stores. In contrast to the strong dependence on calcium mobilization in mesangial cells, calcium entry is reported to play a major role in the contractile response elicited by ANG II in afferent arterioles from rat or rabbit kidneys (8, 14, 15, 29). Nevertheless, a role for calcium mobilization from intracellular sources has also been implicated in responses to ANG II in individual vessel segments and both ANG II and AVP at the whole kidney level (15, 18, 20, 24, 39). Furthermore, there appear to be differences between ANG II effects on afferent and efferent arterioles in terms of calcium signaling pathways, as well as modulation by endothelial factors. Our knowledge of effects of AVP on signal transduction pathways derives largely from whole kidney blood flow studies and cultured glomerular mesangial cells (3, 18, 20, 23).

The purpose of the present study was to evaluate calcium signal transduction mechanisms in a novel preparation of dispersed individual renal vascular SMC obtained from freshly isolated small-diameter preglomerular resistance arterioles. Short vessel segments and unconnected arteriolar SMC were isolated, utilizing an iron oxide-sieving technique, combined with collagenase digestion. With a microscope-based photometer system to quantify fura 2 fluorescence, renal arteriolar SMC were stimulated with ANG II and AVP. Both of these agonists produced a square-shaped, 200- to 300-nM step increase in [Ca²⁺]_i that was immediate and sustained. Pharmacological receptor antagonists demonstrated that the effects of ANG II and AVP were mediated by AT₁ and V₁ receptors, respectively. Prevention of calcium entry by a nominally calcium-free medium [ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)] or by addition of the dihydropyridine, nifedipine, completely prevented or reversed the [Ca²⁺]_i response to either ANG II or AVP, indicating predominant if not exclusive dependence on calcium influx through L-type, voltage-gated calcium entry channels under the experimental conditions.

	METHODS

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Isolation of preglomerular vessels. Experiments were performed in 200- to 230-g male Sprague-Dawley rats obtained from Charles River (Raleigh, NC). To isolate preglomerular vessels, we used an iron oxide-sieving technique previously described by Chatziantoniou and Arendshorst (13). Rats were anesthetized by intraperitoneal injection of pentobarbital sodium, and the aorta was cannulated below the renal arteries through a midline abdominal incision. The aorta was ligated above the renal arteries, the left renal vein was cut, and the kidneys were perfused with ice-cold isotonic saline solution until the effluent was free of blood. Thereafter, the kidneys were perfused with ice-cold magnetized iron oxide suspension (1% Fe₂O₄) for 10-15 s, excised, and placed in a phosphate-buffered saline [PBS (in mM): 17 K₂HPO₄, 3 NaH₂PO₄, 125 NaCl, 5 MgCl₂, at 4°C, pH 7.3].

Segments of preglomerular arterioles were isolated, as previously described for this laboratory (13). Briefly, kidneys were decapsulated, and the cortex was dissected from the medulla. Cortical tissue was placed in a petri dish on top of ice, gently minced with a razor blade, and transferred to a tube containing 5 ml cold PBS. The tissue was homogenized with a Polytron homogenizer at moderate speed (2-3 times for ~5 s). Renal vessels, glomeruli, and surrounding tissue were removed from the crude homogenate with the aid of a magnet. The iron oxide-loaded tissue was resuspended in PBS and passed through needles of decreasing size (22-23 gauge) until the supernatant was free of nonvascular tissue. Thereafter, the vascular suspension was filtered through a 100-µm sieve. The microvessels were recovered from the top of the sieve and resuspended in PBS. This suspension contained microvessels and some tubular elements. A magnet was used to remove the microvessels and separate them from tubular fragments. The remaining vessels consisted mainly of afferent arterioles with some other preglomerular vessels or short pieces of interlobular arteries with diameter <50 µm. More than 95% of the vessels were free of glomerular and tubular tissue. The microvessels were transferred to a tube and incubated with collagenase (1 mg/ml PBS, type 1A, Sigma) for 30 min with constant shaking at 37°C. After collagenase digestion, the tube was shaken vigorously to disperse iron oxide from the vessels; the free iron oxide was removed by a magnet. The remaining solution contained isolated SMC and short pieces of vessels, some of which contained of iron oxide, whereas others were devoid of iron oxide (Fig. 1).

View larger version (109K): [in this window] [in a new window]   Fig. 1.   Light micrographs of freshly isolated smooth muscle cells and segments of preglomerular resistance arterioles <50 µm in diameter. Immunohistological studies revealed that most cells showed positive staining for -actin (a) and heavy chain of myosin SM-1 and SM-2 specific for vascular smooth muscle (b). c and e: Serving as controls for antibody reaction are unstained cells. Photographs of preparations of unstained cells demonstrating individual cells (c) and vessel segments with (d) and without (e) iron oxide. Magnification, ×550.

Measurements of cytosolic calcium concentration. Measurements of [Ca²⁺]_i in renal SMC were performed at room temperature using the calcium-sensitive dye fura 2-acetoxymethyl ester (fura 2-AM, Molecular Probes), as previously described (21, 47). After collagenase treatment, the cells were kept on ice for 15 min before incubation with 4 µM fura 2-AM mixed with 0.02 µM Pluronic acid in PBS for 60 min in the dark at room temperature. Then the solution was centrifuged for 2-3 min, and the fura solution was removed. The pellet of cells was put on ice, and, after 10-15 min, 300 µl of physiological salt solution [PSS (in mM): 135 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 5 D-glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.3] were added slowly back to the cells over 2-3 min. Harvested cells were suspended in calcium-containing PSS for 30 min up to 4 h before fura 2 determinations. The cells were maintained on ice until immediately before an experiment.

For measurements of [Ca²⁺]_i, 10 µl of cell solution were placed on a glass coverslip and centered in the optical field of a ×40 oil-immersion fluorescence objective of an inverted microscope (Olympus IX 70). The cells were excited alternatively with light of 340- and 380-nm wavelengths from a dual-excitation wavelength Delta-Scan equipped with dual monochronometers and a chopper [Photon Technology International (PTI)]. Fluorescence was detected by a photometer after passing signals through a barrier emission filter (510 nm). Fluorescence signal intensity was acquired, stored, and processed by an IBM-compatible Pentium computer and Felix software (PTI). The [Ca²⁺]_i was calculated based on the ratio at 340/380 nm, according to the formula [Ca²⁺]_i = [(R  R_min)/(R_max  R) × (Sf/Sb) × K_d], described by Grynkiewicz et al. (21), using external calibration.

The separate effects of ANG II and AVP mixed in PSS were determined from changes of [Ca²⁺]_i as a function of agonist concentration ranging from 10¹⁰ to 10⁶ M. The responses to each concentration of ANG II or AVP were measured in 6-12 cell preparations. After baseline [Ca²⁺]_i value was recorded for 50 s, 5 µl of ANG II or AVP diluted in PSS were added to the droplet of cells. The [Ca²⁺]_i response was recorded for 100 s. Receptor antagonists for ANG II and AVP were used to determine the main receptor type coupled to calcium signaling. The ANG II (10⁸ M) response mediated by AT₁ receptors was antagonized with losartan (10⁸ M). The V₁ receptor antagonist [d(CH₂)₅,Tyr(NH₂)⁹]AVP was tested at a equimolar concentration of 10⁸ M. Calcium entry was also prevented by utilizing a nominally calcium-free medium, attained by adding EGTA (10⁶ or 10⁸ M). The inhibitory effects of the dihydropyridine, nifedipine (10⁸ M), were used to evaluate the participation of voltage-gated, L-type calcium entry channels. Maintenance of cell position in the optical field was verified in every experiment. Each preparation was tested only once, to avoid possible receptor desensitization or tachyphylaxis.

Immunocytochemistry. Immunocytochemistry was used to detect smooth muscle-specific -actin and smooth muscle-specific myosin heavy chains SM-1 and SM-2, using previously described methods (47). Briefly, several preparations containing freshly isolated SMC were fixed in cold methanol for 5 min. Subsequently, the cells were washed in PBS, blocked with 0.5 M tris(hydroxymethyl)aminomethane (Tris)/Triton containing 2% normal horse serum, and incubated with a monoclonal antibody reacting against -actin specific to smooth muscle cells (Dako, clone 1A4) at a dilution of 1:200 at 4°C overnight (35). A monoclonal antibody diluted 1:600 was used to identify myosin heavy chain SM-1 and SM-2 isoforms (35). The cells were washed three times with Tris/Triton buffer and dehydrated through a series of ethanol washes. Biotinylated antibodies were bound by strepavidin-conjugated peroxidase, and the diaminobenzidine reaction with hydroperoxide was visualized using light microscopy. The employed monoclonal antibody to identify -actin was raised against a synthetic decapeptide having the NH₂-terminal sequence of -smooth muscle actin, which is unique for this isoform. It does not recognize -actin in striated muscle or in myocardium and does not react with - and -cytoplasmic actin in fibroblasts. Owens and associates (35) also have shown that their myosin heavy chain antibody reacts with SM-1 and SM-2 isoforms of SMC and does not react with myosin heavy chains in skeletal muscle or endothelial cells. Cells stained with actin and myosin heavy chain SM isoforms were compared with unstained cells.

Statistical analysis. Data are presented as means ± SE. Data sets were tested with analysis of variance. P < 0.05 was considered statistically significant.

	RESULTS

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The individual SMC of several preparations were examined under light microscopy and were identified by a characteristic elongated, curved or C-shaped appearance (Fig. 1, a-c). Immunohistological studies revealed that almost all cells showed positive staining for -actin (Fig. 1a) and the heavy chains of myosin SM-1 and SM-2 specific for vascular smooth muscle (Fig. 1b). Unstained control cells were negative. The preparations also included short segments of preglomerular arterioles: most contained iron oxide, although some had none (Fig. 1, d and e).

The ability of ANG II and AVP to stimulate [Ca²⁺]_i was assessed using fura 2 fluorescence. A representative tracing of an original recording is shown in Fig. 2. After monitoring the control level of [Ca²⁺]_i, ANG II (10⁸ M) was added to a drop of cells, and the dual-excitation wavelength fluorescence was recorded from 50 to 150 s. The sharp, single step increase in the 340/380 ratio was associated with an increase in signal from the 340 channel and a decrease in 380 channel counts. Thus the [Ca²⁺]_i response was measured as a square-shaped increase that was immediate and sustained throughout the period of ANG II stimulation. Response of each preparation was recorded only once, for ~200 s.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Representative original tracing showing stimulatory effects of angiotensin II (ANG II, 108 M) on cytosolic calcium concentration in one preparation of renal arteriolar smooth muscle cells. A: changes in excitation wavelengths of 340 and 380 nm. B and C: calcium concentration transformed from fluorescence ratio of 340- and 380-nm channels.

Initial experiments determined that ANG II produced similar average [Ca²⁺]_i responses in groups of ~5-20 dispersed individual cells and in single short segments of preglomerular arterioles from the same preparations in a paired fashion. The resting level of [Ca²⁺]_i averaged 209 ± 6 nM in a total of 107 preparations. Figure 3 clearly shows that the basal calcium values did not differ between preparations of dispersed cells and vessel segments. The baseline values averaged 206 ± 9 nM (65 cell preparations) and 215 ± 10 nM (42 vessel segments), respectively (P > 0.5). Figure 3A shows that ANG II (10⁸ M) increased [Ca²⁺]_i from 218 nM to near maximum values at 60 s of 400 ± 48 nM in vessel segments and to 436 ± 65 nM in single SMC; the difference was not significant (P > 0.2). Likewise, the effects of AVP (10⁸ M) on individual cells and short arteriolar segments were similar (Fig. 3B). The paired preparations yielded similar average [Ca²⁺]_i values immediately after stimulation at 60 s (408 in cells vs. 414 nM in segments) and maintained at 150 s (540 vs. 524 nM). Although the [Ca²⁺]_i tended to increase slightly over time, the values at 150 s were not statistically different from those at 60 s (P > 0.2). As baseline values and responses to ANG II and AVP did not differ between single cells and vascular segments, the data in subsequent studies were pooled.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Similarity of responses by single cells and by vessel segments. A: average responses for stimulation of cytosolic calcium concentration by ANG II (108 M) in short segments of preglomerular arterioles (solid line, n = 8) and in individual vascular smooth muscle cells separated from interlobular arteries and afferent arterioles (dashed-dotted line, n = 6). B: mean response of cytosolic calcium concentration to addition of AVP (108 M) at 50 s in single renal arteriolar smooth muscle cells (dashed-dotted line, n = 6) and in short arteriolar segments (solid line, n = 6). Values are means ± SE.

The possible effect of iron oxide on calcium responses was assessed by comparing responses to ANG II (10⁶ M) in short arteriolar segments in two preparations. In arteriolar segments containing iron oxide, the change in [Ca²⁺]_i was 219 ± 18 nM (n = 6), compared with an increase of 207 ± 41 nM (n = 4) in vessels devoid of iron oxide. These changes are not statistically different (P > 0.5). As there were no differences between preparations of mostly single cells compared with isolated vessel segments, either with or without iron oxide, no distinction was made in subsequent experiments, and the results were pooled.

A more comprehensive assessment of the functional properties of the freshly isolated SMC included the ability of multiple concentrations of ANG II or AVP to activate surface receptors and to produce changes in [Ca²⁺]_i. Three different concentrations of ANG II (10¹⁰, 10⁸, and 10⁶ M) were tested. After recording a stable baseline for 50 s, ANG II stimulation caused a rapid increase in [Ca²⁺]_i that reached a maximum value within 5-10 s. The absolute values for [Ca²⁺]_i for the complete recordings are shown in Fig. 4A. Figure 4B shows the maximum [Ca²⁺]_i changes from baseline immediately after initiating hormone stimulation. ANG II caused a dose-dependent increases in [Ca²⁺]_i (Fig. 4B). All concentrations of ANG II produced increases in [Ca²⁺]_i. The maximum [Ca²⁺]_i change from baseline was 221 ± 49 nM in response to ANG II (10⁶ M) (P < 0.01). The peak responses to ANG II to the higher doses (10⁶ and 10⁸ M ) were similar, indicating maximum or saturable stimulation (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Dose-response relation of ANG II effect on cytosolic calcium concentration is freshly isolated rat preglomerular arteriolar vascular smooth muscle cells. A: absolute values for cytosolic calcium concentration before and during stimulation with ANG II at 1010 M (n = 6), 108 M (n = 12), and 106 M (n = 7). B: dose-dependency of average change in cytosolic calcium concentration from baseline (+ P < 0.05, * P < 0.01). Changes in cytosolic calcium concentration after stimulation with 108 and 106 M ANG II are not statistically different. Values are means ± SE.

The temporal response patterns to ANG II stimulation are shown in Fig. 4A. All recordings showed a squarelike pattern with a sharp, initial step rise in calcium followed by subsequent maintenance of the increase. In response to ANG II (10⁸ M), the immediate increase in [Ca²⁺]_i averaged 184 ± 39 nM at 60 s. There was no significant decrease in [Ca²⁺]_i during sustained ANG II stimulation. The [Ca²⁺]_i changes at 150 s (175 ± 34 nM) were similar to those recorded at 60 s.

All tested concentrations of AVP elicited increases in [Ca²⁺]_i. Figure 5A shows that the temporal [Ca²⁺]_i response to AVP was similar to that recorded during ANG II challenge. After a recording a control level, AVP stimulation caused an square-shaped step increase in [Ca²⁺]_i that reached a maximum value within the initial 10 s. The maximal response to AVP (10⁶ M) was an immediate increase of 237 ± 49 nM. The sustained changes in [Ca²⁺]_i elicited by continued exposure to AVP at 150 s did not differ from the immediate response at 60 s: 201 ± 48 and 185 ± 39 nM, respectively. The results in Fig. 5B show AVP effects were dose dependent, with apparent saturation at the high AVP concentrations. The responses to AVP and ANG II stimulation were of similar magnitude.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Vasopressin causes a dose-dependent increase in cytosolic calcium concentration. A: average responses in cytosolic calcium concentration to stimulation with arginine vasopressin (AVP) at 1010 M (n = 6), 108 M (n = 10), and 106 M (n = 6) at 50 s. B: AVP-induced change in cytosolic calcium concentration (+ P < 0.05, * P < 0.01). Changes in cytosolic calcium concentration observed with 108 and 106 M AVP are not significantly different. Values are means ± SE.

To identify the receptor type mediating hormone action of preglomerular arteriolar SMC, additional studies were conducted to evaluate the effect of receptor antagonists on ANG II or AVP-induced increases in [Ca²⁺]_i. Losartan, an AT₁ receptor antagonist, applied in equal concentrations with ANG II (10⁸ M), inhibited ~80% of the response to ANG II (10⁸ M). Figure 6 shows that the [Ca²⁺]_i increase was reduced from 184 ± 39 to 47 ± 27 nM (P < 0.01). In other studies, the effects of AVP were compared without and with the V₁ receptor antagonist (10⁸ M) mixed with AVP (10⁸ M). The data in Fig. 6 demonstrate that the V₁ receptor analog completely inhibited the AVP-induced increase in [Ca²⁺]_i in freshly isolated SMC. The increases in [Ca²⁺]_i recorded at 60 and 150 s did not differ from zero: 13 ± 8 and 19 ± 7 nM, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Inhibition of ANG II effects on cytosolic calcium concentration by AT1 receptor antagonist, losartan. A: absolute calcium concentration following stimulation by ANG II (108 M) (n = 12) and ANG II + losartan (108 M) (n = 6). Inhibitory effect of losartan is statistically significant (P < 0.01). B: vascular V1 receptor antagonist [d(CH2)5,Tyr(NH2)9]AVP inhibits vasopressin effects on cytosolic calcium concentration in freshly isolated afferent arteriolar vascular smooth muscle cells; absolute values for cytosolic calcium concentration after stimulation by AVP 108 M (n = 7) in absence and presence of V1 receptor antagonist (108 M) (n = 6). Change in cytosolic calcium by AVP with V1 present did not differ from zero. Inhibitory effect of the V1 receptor antagonist is statistically significant (P < 0.01). Values are means ± SE.

The importance of calcium entry to calcium signaling in these arteriolar SMC was evaluated by adding EGTA (10⁶ M) to render the external medium nominally calcium free. During continued ANG II (10⁸ M) stimulation, addition of EGTA at 150 s rapidly reduced the ANG II-induced increase in [Ca²⁺]_i to the prestimulation or control level (Fig. 7A). Likewise, in other preparations, EGTA completely reversed the sustained [Ca²⁺]_i response to AVP (10⁶ M) (Fig. 7C). These results establish at strong dependence of the sustained phase on calcium entry from the extracellular compartment.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   A and B: complete reversal of ANG II (108 M) effects on cytosolic calcium concentration by addition of either EGTA (106 M) (A) or nifedipine (108 M ) (B) at 150 s. C and D: summarized results for reversal of AVP (106 M) effects by preventing calcium entry by addition of EGTA (106 M) (C) or of AVP (108 M) by nifedipine (108 M) (D). Calcium concentration quickly returns close to baseline after adding either EGTA or nifedipine. Data are means ± SE for 6 preparations in each group.

In additional experiments, the dihydropyridine nifedipine was used to evaluate the role of L-type, voltage-gated, calcium entry channels in agonist stimulation of renal arteriolar SMC. Nifedipine (10⁸ M) was added to bathing medium after a stimulatory response to ANG II or AVP was recorded. As shown in Fig. 7, B and D, nifedipine application at 150 s promptly reduced the [Ca²⁺]_i to baseline values. Thus nifedipine totally reversed both ANG II and AVP-elicited calcium responses (Fig. 7, B and D).

Other studies were performed to determine whether pretreatment with EGTA or nifedipine would affect resting [Ca²⁺]_i values and whether such pretreatment would affect initiation of the calcium response to either ANG II or AVP. The results in Fig. 8, A and B, top, show that addition of EGTA at 50 s produced consistent, moderate reductions in the resting level of [Ca²⁺]_i: 62 ± 16 nM in ANG II experiments (P < 0.002) and 66 ± 13 nM in AVP studies (P < 0.01). Addition of nifedipine at 50 s had a no discernible effect on basal [Ca²⁺]_i, as the decreases of 17 ± 6 nM for ANG II and 13 ± 5 nM for AVP experiments were not statistically significant (each P > 0.4) (Fig. 8, A and B, bottom). The data in Fig. 8 convincingly demonstrate that in the absence of calcium entry, neither ANG II nor AVP were able to produce an increase in [Ca²⁺]_i. Very small changes in [Ca²⁺]_i were recorded on the average after ANG II (17 ± 6 nM, P > 0.4) or AVP stimulation (11 ± 8 nM, P > 0.5). Thus the initial as well as the sustained calcium responses to agonist receptor activation were dependent on calcium entry through a dihydropyridine-sensitive pathway at a time when there was no discernible change indicative of calcium mobilization from intracellular stores.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effect of addition of EGTA or nifedipine at 50 s on baseline cytosolic calcium concentration in renal afferent arteriolar smooth muscle cells. After pretreatment, neither ANG II (108 M) (A, top and bottom) nor AVP (108 M) (B, top and bottom) caused a demonstrable change in cytosolic calcium concentration when bathing solution was nominally calcium-free (EGTA) or during inhibition of calcium entry with nifedipine. Values are means ± SE for 6-7 preparations in each group.

	DISCUSSION

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The present study provides new information about calcium signaling mechanisms in separated smooth muscle cells freshly isolated from the interlobular artery and afferent arteriole of the rat kidney. A novel aspect of our studies is that the presence and activity of the predominant calcium signaling mechanism were found to be indistinguishable between single arteriolar SMC and SMC in segments of the arterioles. This observation suggests agonist stimulation of calcium entry is largely independent of contact and functional coupling among SMC or interactions with endothelial cells. To characterize our arteriolar SMC preparation, specificity of cell type was demonstrated using antibodies reacting with vascular smooth muscle specific -actin and heavy chains of myosin. The functional coupling of ANG II and AVP receptors to a calcium signaling pathway produced similar dose-related [Ca²⁺]_i responses. A wide concentration range of both ANG II and AVP caused a step- or square-shaped increase in [Ca²⁺]_i that was maintained at near peak levels throughout the observation period. The shape and magnitude of the steady-state [Ca²⁺]_i increases were similar following ANG II or AVP stimulation. Although unique receptors were activated, AT₁ by ANG II and V₁ by AVP, the effector response revealed a common calcium entry mechanism in renal preglomerular arterioles. The peptide-induced changes in [Ca²⁺]_i were prevented by pretreatment with EGTA or nifedipine and were reversed by poststimulation application of either of these agents. The complete prevention and total reversal suggests minimal, if any, participation of calcium mobilization in SMC under the described experimental conditions. Our results demonstrate that calcium influx occurs through voltage-gated, L-type, dihydropyridine-sensitive calcium channels. This is the primary signal transduction pathway following stimulation by either ANG II or AVP in fresh rat preglomerular arteriolar SMC.

Our understanding of intracellular mechanisms that mediate agonist-induced increases in cytosolic calcium concentration and constriction of arterioles in the renal microcirculation is less than complete. In the normal kidney, ANG II and AVP increase total renal vascular resistance by a combination of calcium entry and mobilization (18, 20, 39, 42). Each pathway accounts for approximately one-half of the total vascular response in the kidney as indicated by inhibitory effects of organic calcium channel blockers on calcium entry and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) or heparin on D-myo-inositol 1,4,5-trisphosphate (IP₃)-mediated calcium mobilization and release from internal sources. Segmental arteriolar localization of these effects is under active investigation by several laboratories.

AVP is known to constrict the afferent arteriole in the rat juxtamedullary nephrovascular preparation, but there is a paucity of evidence for the underlying mechanism(s) (22). To our knowledge, there has been only one previous report of AVP action on calcium signaling in isolated SMC of renal arterioles. Inscho et al. (25) presented a typical recording showing that AVP produced a sharp increase in [Ca²⁺]_i that subsequently returned to baseline values by 100 s. Our results indicate that rapid and maintained calcium entry through voltage-gated channels sensitive to nifedipine is the major mechanism mediating the AVP effect. These findings differ from earlier information on AVP-induced calcium mobilization in cultured glomerular mesangial cells (3, 23).

The renal vascular effects of ANG II have been more widely investigated. Results of the present study agree with many earlier studies indicating that calcium entry plays a major role in ANG II-induced contraction and calcium signaling in endothelium-containing segments of afferent arterioles. Our study extends this observation to individual dispersed arteriolar SMC physically separated from each other and endothelial cells (8, 9, 14, 15, 29). Our results also suggest the corollary that calcium mobilization plays a minor role in calcium signaling in isolated SMC from the preglomerular resistance vessels. Based on in vitro changes in vessel diameter, ANG II is thought to contract the afferent arteriole by acting predominantly on voltage-gated calcium channels that are sensitive to the dihydropyridine-class antagonist (8, 15, 29). Moreover, afferent arteriolar [Ca²⁺]_i responses to ANG II are markedly inhibited by removing extracellular calcium or by utilizing calcium channel blockers. Two studies, however, have implicated a combination of calcium mobilization from intracellular stores and calcium entry steps in afferent arteriolar diameter change (15, 24). These studies employed pharmacological agents to putatively inhibit calcium uptake into sarcoplasmic reticulum to partially attenuate ANG II-induced changes in diameter and increases in [Ca²⁺]_i. However, more definitive studies need to test the effects of putative sarcoplasmic Ca²⁺-adenosinetriphosphatase inhibitors in the absence of calcium entry and agents that antagonize the IP₃ receptor. With regard to the efferent arteriole, most of the evidence to date indicates primary calcium entry through channels insensitive to dihydropyridines or calcium mobilization (8, 24, 29). Thus there is a strong likelihood that afferent and efferent arterioles are unique arteriolar segments with different signaling systems and effector responses.

Our results for predominant, if not exclusive, involvement of dihydropyridine-sensitive calcium channels provide no support for the view of calcium release-induced calcium entry and suggest that calcium mobilization is either absent or nonfunctional in calcium signaling in our preparation of freshly isolated vacular SMC. It is not clear why we do not detect a calcium response in a nominally calcium-free medium or in the presence of nifedipine, whereas others investigators have found some involvement of calcium mobilization and uptake by the sarcoplasmic reticulum. This certainly is an important question worthy of more detailed investigation in the future. Such studies are required to determine whether freshly isolated SMC lack or have a masked IP₃-mediated calcium mobilization due to functional regulation or a weak signaling pathway. Alternatively, the requisite calcium stores may have been depleted during the isolation procedure.

It is generally thought that IP₃-mediated calcium release from the sarcoplasmic reticulum serves as a trigger to cause membrane depolarization via activation of potassium or chloride channels, which then activate voltage-sensitive, L-type calcium channels. Our observations advance the interesting notion that calcium entry channels may be activated by a mechanism(s) distinct from prior changes in [Ca²⁺]_i in individual arteriolar SMC in the renal microcirculation. Short-term pretreatment with the dihydropyridine, nifedipine, prevented any [Ca²⁺]_i response to either ANG II or AVP. In addition, antagonism of voltage-gated, L-type channels by nifedipine completely reversed ANG II- or AVP-induced increases in [Ca²⁺]_i. Consistent with these results are our observations that short-term exposure to a nominally calcium-free medium prevented responses to either receptor agonist. Earlier work of other laboratories provides examples that peptide-receptor interaction capable of stimulating calcium entry independent of a calcium mobilization component (36). Our results suggest the presence of a dihydropyridine-sensitive, receptor-operated or receptor-dependent channel. We cannot, however, rule out the possibility that small amounts of intracellular calcium can be redistributed locally to trigger predominant calcium entry. As we have seen with ANG II and AVP, other investigators have noted a total dependence on calcium entry following norepinephrine stimulation in mesenteric resistance arteries, which was completely blocked by an organic calcium channel blocker (10). Calcium entry through a receptor-operated channel may be initiated by receptor activation coupled to a GTP binding protein, inositol phosphates, arachidonic acid metabolites, protein kinase C, or protein tyrosine kinase. Previous work indicates G proteins or IP₃ may directly activate L-type channels (4, 45, 46). Also, protein kinase C may play an important role in phosphorylating, voltage-dependent calcium channels (19, 28, 32). Recent evidence implicates protein tyrosine phosphorylation in ANG II signaling, at least in cultured aortic SMC (31). Another potential mechanism is capacitative-induced calcium entry, in which depleted intracellular pools trigger calcium influx (36).

SMC cultured from aorta or cultured smooth muscle-like glomerular mesangial cells generally exhibit a predominance of intracellular calcium mobilization. Calcium responses to ANG II stimulation in these cultured cells are characterized as rapid, spikelike increases in [Ca²⁺]_i that are short-lived and largely, if not completely, dependent on mobilization of calcium from IP₃-sensitive pools. Accordingly, removal of external calcium has little effect on the ANG response (1-3, 7, 23, 38, 43). Consistent with this general view of calcium mobilization, dantrolene, a putative inhibitor of IP₃-induced release of Ca²⁺ from intracellular pools, inhibits the increase in [Ca²⁺]_i elicited by ANG II and AVP (43). Nevertheless, some studies suggest the presence of and a functional role for calcium entry through dihydropyridine-sensitive channels in cultured SMC and mesangial cells. The degree of involvement varies considerably among studies and preparations but overall tends to represent <25-50% of the calcium response. Several investigators report that ANG II produces a sustained, moderate increase in [Ca²⁺]_i that is dependent, in part, on the presence of external calcium (7, 9, 43, 47). In some conditions and preparations of cultured aortic SMC, calcium entry may take place through channels that are insensitive to dihydropyridine agents (11, 48).

The baseline calcium concentration of ~200 nM in our isolated interlobular arterial and afferent arteriolar SMC is somewhat higher than the 50-120 nM observed in the cultured SMC from rat resistance vessels and cultured mesangial cells (3, 43, 47). Our values are also higher than those reported by Inscho et al. (25) for freshly isolated preglomerular arteriolar SMC. A portion of the basal [Ca²⁺]_i in our preparations was maintained by calcium entry that was dependent on removal of extracellular calcium but unaffected on the average by nifedipine. Previous studies on isolated renal arteriolar vessel segments report baseline [Ca²⁺]_i values that vary between 130 and 200 nM, with a small but consistent tendency to be higher in freshly isolated SMC compared with cultured SMC (9, 15). Due to a general similarity among different preparations involving microdissection, sieving, and the juxtamedullary nephrovascular preparation, we think it unlikely that our method of isolation differs markedly from those used by others. We find no evidence that the baseline values and calcium responses to agonist stimulation are influenced by our cell/vessel isolation method utilizing iron oxide and sieving. The present results indicate that a vessel segment responds in similar fashion to ANG II whether or not iron oxide is present in the lumen of the vessel. Also, our single cells yielded results qualitatively and quantitatively similar to those obtain on short vessels segments. The sustained changes we observed in [Ca²⁺]_i are similar in magnitude to the peak responses reported by Inscho et al. (25) for freshly-isolated preglomerular arteriolar SMC. However, the general pattern of the calcium responses differed. We observed that both ANG II and AVP elicited [Ca²⁺]_i increases that were sustained close to the level of the immediate response. In contrast, Inscho et al. (25) found that continuous ANG II stimulation for 300 s produced a biphasic response with [Ca²⁺]_i stabilizing at ~50% of the initial peak. The immediate peak response to AVP, on the other hand, decayed to baseline during 50 s of continued AVP stimulation. The ANG II- and AVP-induced changes in [Ca²⁺]_i we observed were more robust than previously published for freshly harvested preglomerular vessel segments (15, 27). Previous reports on isolated rat afferent arteriolar segments indicate that 10⁸ M ANG II causes a square-shaped response, characterized by a rapid and sustained [Ca²⁺]_i increase of ~100 nM (15). A qualitatively different temporal response is reported for microdissected rabbit afferent arterioles. ANG II causes an immediate, relatively weak 20 nM increase in [Ca²⁺]_i that returns to the baseline level by 100 s (27).

The calcium response to both peptides in our studies was inhibited by their selective receptor antagonist. The AT₁ receptor antagonist losartan inhibited of the ANG II-induced change in [Ca²⁺]_i by up to 75-80%, which agrees well with the amount of antagonism of ANG II binding to freshly isolated preglomerular resistance arterioles and of renal vasoconstriction produced by ANG II in vivo (13). Other groups have found that losartan antagonizes the calcium response to ANG II in cultured rat preglomerular arteriolar SMC and mesangial cells (16, 47). With regard to the action of AVP on [Ca²⁺]_i, we found that a V₁ receptor antagonist produced complete inhibition in our preglomerular arteriolar SMC, confirming purity of the vascular preparation, which is essentially devoid of tubular epithelial cells. Previous studies have shown the V₁ receptor is the major type present in cultured SMC from the rat and human aorta and cultured rat mesangial cells, and we have found that virtually all of AVP-induced renal vasoconstriction in vivo in the rat is mediated by the V₁ receptor (3, 13, 17, 41, 43, 44).

In conclusion, our study provides new data for freshly isolated SMC from interlobular arteries and afferent arterioles demonstrating that a receptor-operated or receptor-dependent mechanism increases [Ca²⁺]_i after stimulation of AT₁ receptors by ANG II or V₁ receptors by AVP. Agonist-induced increases in [Ca²⁺]_i were prevented and reversed by a nominally calcium-free external medium and by the dihydropyridine, nifedipine. The calcium responses to ANG II and AVP were indistinguishable between single dispersed SMC and short segments of preglomerular arterioles. This was the case for both the overall shape of the response and the magnitude of the immediate and sustained changes in [Ca²⁺]_i. This observation suggests that communication between cells within small segments of isolated vessels plays a minor role in the qualitative and quantitative characteristics of AT₁- and V₁-mediated stimulation of [Ca²⁺]_i under the experimental conditions. We found no evidence for major participation of calcium mobilization from internal reserves in our freshly isolated renal SMC. Both the immediate and sustained [Ca²⁺]_i responses are predominately mediated by a calcium entry mechanism involving L-type, voltage-operated, dihydropyridine-sensitive channels in our freshly harvested smooth muscle cells from renal resistance vessels.