(Hypertension. 1998;31:421.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Calcium Mobilization Contributes to Pressure-Mediated Afferent Arteriolar Vasoconstriction
Department of Physiology (E.W.I., A.K.C., V.M., J.D.I,), Tulane University School of Medicine, New Orleans, Louisiana 70112.
Correspondence to Edward W. Inscho, Department of Physiology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112. E-mail einscho{at}mailhost.tcs.tulane.edu
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Abstract |
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Preglomerular responses to vasoactive agonists utilize calcium released from intracellular stores and activation of calcium influx pathways to elicit vasoconstriction. The current study was performed to determine the role of calcium release from intracellular stores on the afferent arteriolar response to increases in perfusion pressure. Experiments were performed, in vitro, using the blood perfused, juxtamedullary nephron technique combined with videomicroscopy. The response of afferent arterioles to 30 mm Hg increases in perfusion pressure was determined before and after depletion of intracellular calcium pools with a 10-minute preincubation with 1 µmol/L thapsigargin or 100 µmol/L cyclopiazonic acid. Afferent arteriolar diameter averaged 20.2±1.0 µm (n = 19) at a control perfusion pressure of 100 mm Hg. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent caliber by 10.7±1.0% (P<.05 versus con) and by 24.7±1.6% (P<.05 versus diameter at 130 mm Hg); respectively. Thapsigargin significantly increased afferent diameter by 21±2% (n = 6) at 100 mm Hg and prevented pressure-induced autoregulatory responses. Afferent diameter averaged 24.3±1.7, 24.5±1.8 and 24.3±1.8µm at perfusion pressures of 100, 130 and 160 mm Hg; respectively. Cyclopiazonic acid treatment also inhibited autoregulatory behavior but did not alter resting vessel diameter. Afferent arteriolar diameter (n = 6) averaged 21.4±1.9µm at 100 mm Hg and 20.9±2.1 and 20.5±2.2µm at 130 and 160 mm Hg; respectively. Additional studies were performed to assess the role of phospholipase C activity in pressure-mediated autoregulatory behavior of afferent arterioles. Step increases in perfusion pressure decreased afferent diameter by 10.7±3.8 and 21.7±4.1%; respectively. Administration of the phospholipase C inhibitor, U-73122, (5 µmoles/L) did not significantly alter baseline diameter but did attenuate the pressure-mediated vasoconstrictor response. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent diameter by only 6.5±1.5 and 10.0±2.0%; respectively. These data demonstrate that interruption of calcium mobilization with thapsigargin, cyclopiazonic acid, or phospholipase C inhibition markedly attenuates pressure-mediated afferent arteriolar vasoconstriction and suggests that autoregulatory adjustments in afferent arteriolar diameter involve calcium release from inositoltrisphosphate (IP3)-sensitive intracellular stores.
Key Words: calcium mobilization • thapsigargin • cyclopiazonic acid • autoregulation • phospholipase C • U-73122 • microcirculation • renal
Abbreviations: TGF = tubuloglomerular feedback • IP3 = inositoltrisphosphate
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Introduction |
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One of the main functions of the preglomerular microvasculature is to regulate glomerular capillary pressure by adjusting afferent arteriolar resistance in response to changes in perfusion pressure.1–14 This autoregulatory behavior represents the combined influences of both myogenic and tubuloglomerular feedback (TGF) generated alterations in microvascular wall tension regulating preglomerular resistance.2,9 While some aspects of autoregulation have been delineated, the intracellular signal transduction events responsible for pressure-dependent adjustments in preglomerular resistance remain poorly understood. Previous studies have shown that vascular resistance changes evoked in response to alterations in renal perfusion pressure primarily reflect adjustments in afferent arteriolar diameter.1,4,5,11,13,15,16 Elevation of cytosolic calcium concentration represents an important component in the afferent arteriolar response to perfusion pressure changes.1,6,9,11,12,14–19 Pressure-mediated vasoconstriction of afferent arterioles relies heavily of the influx of extracellular calcium through voltagegated calcium channels;1,6,9,11,12,14–16,18,19 although the nature of the depolarizing stimulus remains to be identified.20,21 Also unclear is the role of calcium release from intracellular stores in the vascular response to mechanical stimulation.20,22,23
Pressure-mediated elevations of cytosolic calcium concentration in vascular smooth muscle can occur through activation of calcium influx pathways involving stretch-activated, nonselective cation channels; membrane depolarization and activation of potential dependent ion channels; and/or through mobilization of calcium release from intracellular pools.24–32 Studies in nonrenal vascular preparations have shown that mechanical stimuli evoke rapid increases in cytosolic calcium concentration.12,25–29 Although still controversial, some studies suggest that part of the increase in cytosolic calcium concentration derives from calcium released from intracellular pools.22,27,29,30,33,34 Other studies have shown that increases in transmural pressure stimulate phospholipase C and phospholipase D activity leading to generation of IP3 and diacylglycerol, whereas others implicate a role for pressure-mediated activation of protein kinase C.23,29,31,33 Finally, it has recently been reported that rapid stretching of individual vascular smooth muscle cells stimulates a rapid increase in cytosolic calcium concentration partly through release of calcium from intracellular stores.27 Thus, pressure-mediated calcium release from intracellular stores may represent an important step in the sequence of events coupling changes in transmural pressure and the subsequent myogenic vasoconstriction shown to occur in vascular smooth muscle from most vascular beds.
With the recent development of agents which selectively perturb the filling state of intracellular calcium pools, it is now possible to manipulate these pools and determine the influence of calcium store integrity on microvascular responsiveness to acute changes in perfusion pressure.35–39 In addition, new enzyme inhibitors selective for steps of the intracellular calcium response pathway23,40 allow interruption of specific components of the phospholipase C/ calcium release cascade in order to determine their role in mediating pressure-dependent renal microvascular responses.
The current studies were performed to determine the effect of calcium store depletion on the afferent arteriolar response to acute changes in perfusion pressure. Direct assessment of afferent arteriolar responsiveness to step increases in perfusion pressure was performed before and during depletion of intracellular calcium pools with the Ca2+ ATPase inhibitors, thapsigargin and cyclopiazonic acid. Thapsigargin and cyclopiazonic acid have been shown to deplete IP3-sensitive intracellular calcium stores, thereby interrupting IP3-dependent, agonist-mediated responses.35–39 In addition, studies were performed to determine the effect of phospholipase C inhibition, with U-73122, on the afferent arteriolar response to increases in perfusion pressure.
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Materials
Enalaprilat was a gift from Merck Sharp and Dohme. NE (Levophed) was obtained from Winthrop Pharmaceuticals. U-73122 was purchased from BIOMOL Research Laboratories Inc. Thapsigargin was purchased from Research Biochemicals Inc. Cyclopiazonic acid and all other reagents were purchased from Sigma Chemical Company.
Kidney Preparation
Studies were performed as approved by the Tulane University Advisory Committee for Animal Resources. Experiments were conducted in vitro using the blood perfused juxtamedullary nephron technique as previously described.1,41,42 For each experiment, two male Sprague-Dawley rats (350 to 400 g) were anesthetized with sodium pentobarbital (40 mg/kg; IP) and pretreated (30 minutes) with the converting enzyme inhibitor, enalaprilat (2 mg; IV). Perfusate blood was collected from the kidney and blood donors and was prepared as previously described.1,42 The blood donor rat was nephrectomized and, 30 minutes later, exsanguinated into a syringe containing heparin (500 units). The plasma and erythrocyte fractions were separated from the centrifuged blood and the leukocyte fraction was discarded. The plasma was filtered (0.2 µm) before being mixed with erythrocytes to yield a hematocrit of approximately 0.33. The reconstituted blood was filtered through a 5 µm nylon mesh.
The right renal artery of the kidney donor was cannulated, and the kidney was immediately perfused with a Tyrode’s buffer solution containing 52.0 g/L bovine serum albumin and a complement of L-amino acids.1,42 The perfused kidney was removed and sectioned longitudinally to leave the papilla intact on the dorsal two-thirds portion of the kidney. The papilla was reflected back, and the pelvic mucosa was removed to expose the main arterial branches, renal tubules, glomeruli, and related microvasculature of juxtamedullary nephrons. Ligation of the terminal ends of the large arteries restored perfusion pressure to the perfused cortical and papillary tissue. When the dissection was completed, the cell-free perfusate was replaced with the reconstituted, oxygenated blood. Perfusion pressure was monitored at the cannula tip and was set at 100 mm Hg. The inner cortical surface of the kidney was continuously superfused with warmed (37°C) Tyrode’s buffer containing 10.0 g/L bovine serum albumin, and the kidney was allowed to equilibrate for at least 15 minutes.
The perfusion chamber containing the prepared kidney was affixed to the stage of a Nikon Xenophot microscope (Nikon Corp) equipped with long working distance objectives. Video images, generated with a high resolution Newvicon camera (NC-67m, Dage-MTI) were electronically enhanced (image processor MFJ-1425, MFJ Enterprises Inc) and displayed on a video monitor while being simultaneously recorded on videotape for analysis. Afferent arteriolar inside diameters were measured at a single site using an image shearing monitor (Model 901, Instrumentation for Physiology and Medicine) calibrated with a stage micrometer. The steady-state diameter of each arteriole was calculated for each experimental period by averaging the measurements taken during the last 1 or 2 minutes of that period. Afferent arterioles were selected for study based on the clarity of the vascular walls and the adequacy of blood flow through the vessel lumen.
Autoregulation Protocols
Effect of Thapsigargin or Cyclopiazonic Acid on the Afferent Arteriolar Response to Increasing Perfusion Pressure
The effect of increasing perfusion pressure on afferent arteriolar diameter was determined before and after calcium pool depletion with either thapsigargin or cyclopiazonic acid. Thapsigargin is a sesquiterpene lactone compound, which has been shown to specifically inhibit Ca2+-ATPase from the endo- and sarcoplasmic reticulum.35,36,43 Cyclopiazonic acid is an indole tetramic acid, produced by Aspergillus or Penicillium, which has also been shown to be a selective inhibitor of sarcoplasmic reticulum Ca2+-ATPase.38,43,44 The thapsigargin (1 µM) and cyclopiazonic acid (100 µM) concentrations were chosen based on previous studies demonstrating that these concentrations were maximally effective in transiently increasing cytosolic calcium coincident with intracellular store depletion.37 In addition, previous studies have shown that 1 µM thapsigargin markedly attenuated afferent and efferent arteriolar responsiveness to angiotensin II42 and norepinephrine.42,45
The following autoregulatory experiments were performed. Afterent arteriolar diameter was determined during the 5-minute control period with perfusion pressure set at 100 mm Hg. Subsequently, perfusion pressure was increased in 30 mm Hg increments, to 130 mm Hg and 160 mm Hg, in consecutive 3-minute periods. Arteriolar diameter was monitored constantly during each pressure step, and the steady-state diameter was determined by computing the mean diameter during the final minute. Finally, perfusion pressure was returned to 100 mm Hg and a 5-minute recovery period ensued. The role of intracellular calcium stores in the afferent arteriolar response to increasing perfusion pressure was assessed by treating each arteriole with thapsigargin (1 µM) or cyclopiazonic acid (100 µM) for 10 minutes before increasing perfusion pressure again to 130 mm Hg and 160 mm Hg. Perfusion pressure was then returned to 100 mm Hg for a 5-minute recovery period.
Effect of the Phospholipase C inhibitor, U73122 on the Afferent Arteriolar Response to Increasing Perfusion Pressure
A second series of experiments was performed to determine the role of phospholipase C in the afferent arteriolar response to increasing perfusion pressure. For these experiments, afferent arteriolar diameter was determined during the 5-minute control period and during the subsequent 3-minute periods at 130 mm Hg and 160 mm Hg; respectively. Following the 5-minute recovery period, the phospholipase C inhibitor, U73122 (5µmoles/L), was administered via the superfusion solution, and the arterioles were allowed to equilibrate for a minimum of 20 minutes. Following the equilibration period, afferent arteriolar diameter was again measured at perfusion pressures of 100 mm Hg, 130 mm Hg, and 160 mm Hg before the final recovery period. As previously stated, vessel diameter was monitored continuously during the entire protocol.
Statistical Analysis
Statistical comparisons within each series were made using a one-way analysis of variance for repeated measures combined with the Newman Keuls multiple range test. A probability value of P<.05 was considered significant. All data are reported as the mean±SE.
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A total of 19 juxtamedullary afferent arterioles were used for these studies. These arterioles averaged 454±30µm in length and 20.2±1.0µm in diameter. Vascular diameter measurements were made an average of 220±15µm from the glomerulus representing the midpoint of the vessel length. There was no significant difference between the resting afferent arteriolar diameter, length, or measurement distance from the glomerulus between any of the treatment groups.
Thapsigargin Studies
The effect of thapsigargin on the afferent arteriolar response to increasing perfusion pressure is shown in Fig 1. Six afferent arterioles averaged 20.0±1.2 µm during the control period and this diameter decreased reversibly in response to consecutive 30 mm Hg increases in perfusion pressure (Fig 1A). Afferent caliber decreased significantly to 18.0±1.2 and 15.6±1.2 µm at perfusion pressures of 130 mm Hg and 160 mm Hg; respectively. This represents a decline in afferent diameter to 90±1% and 78±2% of control; respectively (Fig 1B). Returning perfusion pressure to 100 mm Hg resulted in a complete reversal of the pressure-mediated vasoconstriction. Thapsigargin treatment resulted in a significant relaxation of these afferent arterioles with vessel diameter increasing by 21±2% and abolished the pressure-dependent vasoconstriction observed under control conditions. Afferent arteriolar diameter averaged 24.3±1.7, 24.5±1.8, 24.3±1.8 µm at perfusion pressures of 100 mm Hg, 130 mm Hg, and 160 mm Hg; respectively, corresponding to 101±1% and 100±1% of the diameter with thapsigargin alone (Fig 1B).
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= P<.05 versus thapsigargin treatment.
Cyclopiazonic Acid Studies
The effect of cyclopiazonic acid on the afferent arteriolar response to perfusion pressure was also examined and the results of those studies are illustrated in Fig 2. The diameter of six afferent arterioles averaged 21.4±2.1 µm during the control period, and this diameter decreased in a pressure-dependent manner in response to consecutive step increases in perfusion pressure to 130 mm Hg and 160 mm Hg. Afferent caliber decreased significantly to 19.1±2.1 and 15.6±1.6 µm, respectively. Cyclopiazonic acid treatment transiently vasoconstricted the afferent arterioles. Vessel diameter decreased to a minimum diameter of 19.7±2.1 µm within approximately 1 minute of exposure before gradually recovering to a diameter similar to control (21.4±2.0 µm). In the continued presence of cyclopiazonic acid, the afferent arteriolar diameter response to increasing perfusion pressure was reassessed. Whereas increasing perfusion pressure 130 mm Hg and 160 mm Hg reduced afferent diameter to 89±2% and 73±2% of control under the control conditions (Fig 2B), in the presence of cyclopiazonic acid, afferent caliber remained essentially unchanged. Afferent diameter averaged 21.4±2.1 and 20.5±2.2 µm at perfusion pressures of 130 mm Hg and 160 mm Hg; respectively. These values represent diameters of 98±1% and 95±2% of the diameter with cyclopiazonic acid alone (Fig 2B).
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U-73122 Studies
Studies were also performed to assess the role of phospholipase C activity in pressure-mediated afferent arteriolar autoregulatory behavior. These studies were performed by assessing pressure-dependent changes in afferent arteriolar diameter before and during administration of the phospholipase C inhibitor, U-73122. The results of those experiments are illustrated in Fig 3. The control diameter of 7 afferent arterioles averaged 20.5±.9 µm (Fig 3A). Increasing perfusion pressure in two 30 mm Hg increments decreased vessel diameter to 89±4% and 78±4% of the control diameter (Fig 3A and 3B). Arteriolar diameter returned to the control diameter (20.5±0.9) when perfusion pressure was reset to 100 mm Hg. Administration of 5 µmoles/L U-73122 did not significantly alter baseline afferent arteriolar diameter. With perfusion pressure set at 100 mm Hg, afferent diameter averaged 19.9±.7 µm which is 99±3% of the starting diameter. In the presence of U-73122, the pressure-mediated afferent arteriolar vasoconstrictor response was reassessed (Fig 3A and 3B). Increasing perfusion pressure to 130 mm Hg and 160 mm Hg reduced afferent diameter significantly to 94±2% and 90±2% of the baseline diameter at 100 mm Hg; however, as shown in Fig 3B, the magnitude of the response at 160 mm Hg was significantly attenuated, compared to the control response.
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Discussion |
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In the present study, we directly examined the effect of intracellular calcium store depletion and phospholipase C inhibition on the afferent arteriolar response to increases in perfusion pressure. The results of these studies demonstrate that pressure-mediated autoregulatory adjustments in juxtamedullary afferent arteriolar diameter involve release of Ca2+ from purported IP3-sensitive, intracellular calcium stores. Consistent with this observation is the finding that pressure-mediated adjustments in afferent arteriolar diameter are adversely affected by inhibition of phospholipase C activity. Therefore, phospholipase C activation with the subsequent generation of IP3 appears to be an important component in the sequence of events culminating in autoregulatory adjustments in renal vascular resistance.
The kidney is capable of precisely regulating renal blood flow and glomerular filtration rate over a wide range of renal perfusion pressures.2,4,9 Exactly how renal autoregulation is accomplished has been the focus of intensive investigation, yet many aspects of the phenomenon remain to be explained. Nevertheless, several components of the autoregulatory response have been resolved. There is a general consensus that whole kidney autoregulatory behavior is a manifestation of the intrinsic myogenic behavior of the microvasculature supplemented by the vasoregulatory influences of the TGF mechanism of the juxtaglomerular apparatus.2,4,9 Direct evidence for the contribution of myogenic influences in the renal microvascular autoregulatory response comes from studies performed in the hydronephrotic kidney preparation, which maintains an intact renal microvascular network in the absence of renal tubules or juxtaglomerular apparatus,11,13–15 microdissected afferent arterioles,3 or preparations in which TGF-dependent responses have been inhibited.1,8 Evidence for the contribution of the TGF component comes from micropuncture studies where changes in distal tubular perfusion result in changes in preglomerular resistance without any change in renal perfusion pressure9,18,46,47 and from isolated kidney preparations where loop perfusion and macula densa function can be manipulated.1,8,16 Myogenic vasoconstriction purportedly involves activation of stretch-activated, nonselective cation channels followed by depolarization and activation of voltage-gated calcium channels.14,21,24–30 Both TGF-mediated and myogenic adjustments in afferent arteriolar resistance can be blocked by L-type calcium channel antagonists thus attesting the crucial role voltage-dependent calcium influx plays in the autoregulatory response.1,6,14,15,18,20,46
The role of calcium release from intracellular stores is more controversial. Earlier studies suggested that calcium mobilization from intracellular stores is not a major contributor to whole kidney autoregulatory responses in the dog.22 However, studies performed in other tissues exhibiting autoregulatory behavior suggest that calcium mobilization from intracellular stores may represent an important component in the overall response.26,27,29 D’Angelo and Meininger presented a flow scheme describing the critical elements involved in myogenic responses of vascular smooth muscle.29 Accordingly, vascular smooth muscle responses to mechanical stretch include G-protein-dependent activation of phospholipase C with the subsequent generation of IP3 leading to calcium mobilization from IP3-sensitive intracellular calcium stores. More recently, Davis and coworkers reported that mechanical stretch of isolated coronary artery smooth muscle cells leads to the release of stored calcium into the cytosol.27 While this observation supports a role for calcium mobilization as part of the myogenic response to stretch, similar observations have not been made from small caliber arterioles (<20 µm) or from renal microvascular smooth muscle cells.
Thapsigargin and cyclopiazonic acid inhibit the activity of the sarcoplasmic reticulum Ca2+ ATPase.35,36,38,43,44 Calcium ATPase inhibition results in depletion of intracellular calcium stores as they are emptied via endogenous leak pathways without being replenished through the activity of the sarcoplasmic reticulum Ca2+ ATPase.37 This eliminates the availability of a readily mobilizable calcium repository to be accessed by store-dependent vasoactive stimuli even in the presence of normal extracellular Ca2+ concentrations. We have previously shown that thapsigargin treatment markedly attenuated the vasoconstriction of juxtamedullary afferent arterioles induced by angiotensin II or norepinephrine.42 In the current report, both thapsigargin and cyclopiazonic acid markedly attenuated the afferent arteriolar vasoconstriction-induced by acute increases in perfusion pressure. This suggests that pressure-mediated calcium release from intracellular stores may be an essential step in the sequence of events coupling the increase in transmural pressure to the subsequent autoregulatory vasoconstriction.
Thapsigargin and cyclopiazonic acid have been used extensively as Ca2+ ATPase inhibitors for the depletion of intracellular calcium stores in a broad range of tissues and cell types.35,37,38,43,44,48,49 Nevertheless, there are reports that these agents may have other effects that may complicate interpretation of data obtained using them.50–54 Studies have suggested that thapsigargin and cyclopiazonic acid may also stimulate Ca2+ influx and directly cause vasoconstriction by elevating cytosolic calcium and causing membrane depolarization. In vascular smooth muscle, calcium influx is reported to occur through activation of L-type calcium channels as well as stimulating another dihydropyridine-insensitive pathway.51,54 Other studies have suggested that use of these agents results in drug-induced relaxation through inhibition of voltage-activated calcium channels50–53 or through activation of Ca2+-activated K+ channels49,50 following an elevation of cytosolic calcium concentration. Nevertheless, the possibility that such nonspecific effects may have invalidated or confounded our findings in the current report is unlikely. Both thapsigargin and cyclopiazonic acid completely eliminated pressure-mediated vasoconstriction of afferent arterioles. For two structurally dissimilar compounds to alter pressure-mediated autoregulatory behavior through an identical nonspecific effect is doubtful especially when qualitatively similar results are obtained by inhibiting phospholipase C activity.
The possibility that three interventions designed to reduce calcium mobilization from intracellular stores would all nonspecifically compromise autoregulatory behavior is improbable. Furthermore, the actions of these Ca2+ ATPase inhibitors are not totally consistent with the reported nonspecific actions for these agents. For example, if thapsigargin was interfering with calcium influx through voltage-gated calcium channels, it would result in attenuation or blockade of pressure-mediated autoregulatory adjustments in afferent arteriolar diameter. However, we have previously demonstrated that 1 µmole/L thapsigargin does not inhibit the afferent arteriolar vasoconstriction induced by membrane depolarization with 55 mmoles/L KCl.42 Similarly, Bay K 8644, has been shown to vasoconstrict afferent arterioles in the presence of cyclopiazonic acid.55 Retention of KCl-mediated and Bay K 8644-mediated afferent arteriolar vasoconstriction in the presence of thapsigargin or cyclopiazonic acid, respectively, argues against the nonspecific interaction of these agents with L-type calcium channels. In the present study, thapsigargin was found to increase afferent arteriolar diameter. This vasodilation is inconsistent with activation of Ca2+ influx. Cyclopiazonic acid had an identical effect on the autoregulatory response compared to thapsigargin but did not alter resting afferent arteriolar diameter. Therefore, in the presence of these Ca2+ ATPase inhibitors, the ability of calcium channels to alter afferent arteriolar caliber is retained.
Finally, Ca2+-dependent activation of K+ channels with the subsequent hyperpolarization of afferent arteriolar smooth muscle is also unlikely, since this would lead to vasorelaxation. In the current report, only thapsigargin had a significant effect on resting afferent arteriolar diameter. Thapsigargin treatment increased vascular diameter, whereas afferent diameter was unchanged in the presence of cyclopiazonic acid and tended to decrease slightly during inhibition of phospholipase C. It is doubtful that all three agents could be inhibiting autoregulatory behavior by activation of Ca2+-dependent K+ channels while each agent exerts a different effect on resting afferent arteriolar diameter. Accordingly, rather than nonspecific effects being responsible for the results obtained, these data strongly suggest that direct inhibition of the sarcoplasmic reticulum Ca2+ ATPase by thapsigargin and cyclopiazonic acid eliminated a crucial intracellular calcium source required to sustain the sequence of signal transduction events leading to an appropriate pressure-dependent vasoconstrictor response. Therefore, the data presented here support the hypothesis that thapsigargin and cyclopiazonic acid treatment depletes important afferent arteriolar calcium stores and interrupts a pivotal component of the signal transduction cascade induced by acute changes in perfusion pressure.
If changes in perfusion pressure and thus changes in microvascular transmural pressure stimulate autoregulatory responses involving calcium mobilization from IP3-sensitive storage pools, then this response should involve activation of phospholipase C. Therefore, inhibition of phospholipase C activity should attenuate, or abolish, the microvascular response to increases in perfusion pressure. The results in the current report are consistent with this hypothesis. Phospholipase C inhibition, with U-73122, significantly attenuated the afferent arteriolar response to increased perfusion pressure. The magnitude of the attenuation appears to be smaller than the response obtained with thapsigargin or cyclopiazonic acid, and it may reflect incomplete blockade of phospholipase C activity or it may indicate that other second messenger sources or pathways also participate in the mobilization of intracellular Ca2+, such as cyclic ADP-ribose or tyrosine kinase.56 Nevertheless, attenuation of IP3 formation by the phospholipase C inhibitor, U-73122 resulted in a clear attenuation of the pressure-dependent autoregulatory response of juxtamedullary afferent arterioles.
As previously stated, whole kidney autoregulation occurs through the combined influences of both TGF and myogenic regulation of microvascular function.2,4,9 The results of the current study do not allow us to identify which of these two regulatory systems have been manipulated by these pharmacological agents. Evidence suggests that the TGF mechanism exerts the greatest influence on preglomerular resistance along portions of the afferent arteriole nearest to the glomerulus; whereas myogenic regulation is most prominent at sites more distant from the glomerulus.1,11,13,14 In this report, afferent arteriolar diameter measurements were obtained near the midpoint (49±1%) of the vessel length. Evidence suggests that this region is subject to regulation by both myogenic and TGF signals.7,13,14 Nevertheless, it is interesting to note that despite the absence of pressure-mediated vasoconstriction during thapsigargin, cyclopiazonic acid or U-73122 treatment, these afferent arterioles did maintain a relatively stable luminal diameter. Therefore, each increment in perfusion pressure still elicited an increase in vascular wall tension sufficient to prevent the arteriolar caliber from increasing passively with each pressure step, yet insufficient to reduce luminal diameter below control. Retention of some tension generating capacity in response to increases in perfusion pressure suggests that inhibition of calcium release from intracellular stores may primarily impact on just one of the two branches of overall renal autoregulatory behavior. This is supported by the studies focusing on the role of voltage-dependent calcium channels in autoregulation. Voltage-gated calcium channels play a major role in both TGF and myogenic autoregulatory adjustments in preglomerular resistance.1,6,13–16,18–20,46 In the presence of L-type Ca2+ channel antagonists, step increases in perfusion pressure coincide with passive, pressure-dependent increases in arteriolar diameter.1,6,14–16,19,20 The ability of calcium channel blockers to result in a passive afferent arteriolar diameter relationship with alterations in perfusion pressure is largely a reflection of the important role L-type calcium channels play in the regulation of renal microvascular function and responsiveness. Calcium channel inhibition blocks a major Ca2+ influx pathway and eliminates one of the primary mechanisms for supporting a sustained elevation of cytosolic Ca2+ concentration, and it eliminates a potentially important source of Ca2+ for the refilling of intracellular calcium stores. Therefore, the complete loss of autoregulatory behavior during calcium channel blockade, as reflected by the passive behavior of afferent arteriolar diameter in response to changes in perfusion pressure, could reflect the combined impact of direct inhibition of an important calcium influx pathway and the indirect depletion of intracellular stores by reducing cytosolic Ca2+ concentration and impeding replenishment of intracellular Ca2+ stores. Since the arterioles examined in the current report do not behave passively by increasing luminal diameter in response to increased luminal pressure, one must conclude that these arterioles retain some functional autoregulatory capacity.
We cannot discount the possibility that thapsigargin, cyclopiazonic acid, or U-73122 may be directly interfering with macula densa function. Given that the macula densa plays an important role in TGF-mediated regulation of preglomerular microvascular function and that the macula densa and afferent arteriolar vascular smooth muscle are near the cortical surface and lie in close proximity to one another, it is possible that these pharmacological agents are directly influencing macula densa function. Accordingly, the results presented in this report could suggest that inhibition of intracellular calcium mobilization in macula densa cells with thapsigargin, cyclopiazonic acid, and U-73122 attenuates or abolishes TGF-dependent responses. This conclusion is supported by earlier micropuncture studies evaluating the role of calcium in the transmission of TGF signals.46,47 Bell and Reddington demonstrated that retrograde perfusion of the macula densa region of the distal nephron elicited significant reductions in stop flow pressure.47 These stop flow pressure responses were not affected by alterations of perfusate Ca2+ concentration, or by calcium channel blockade, thus suggesting that calcium influx across the luminal membrane of macula densa cells is not an essential step in activation of TGF signals. In contrast, when the macula densa region was exposed to a perfusate containing 8-(N, N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative inhibitor of calcium release from intracellular storage pools, stop flow pressure responses were markedly attenuated. Thus, calcium release may play a significant role in coupling macula densa function with changes in preglomerular resistance and may support the contention that thapsigargin, cyclopiazonic acid, and phospholipase C inhibition could be inhibiting TGF-mediated autoregulatory adjustments in afferent arteriolar diameter through direct actions on the macula densa. They do not, however, allow differentiation of the extent of involvement of calcium mobilization in myogenic versus TGF-dependent autoregulatory responses. Further investigation of this possibility requires that pressure-mediated afferent arteriolar responses be assessed under feedback-dependent and -independent conditions.
In summary, depletion of intracellular calcium stores with thapsigargin or cyclopiazonic acid markedly attenuates juxtamedullary afferent arteriolar vasoconstrictor responses induced by acute increases in perfusion pressure. In addition, inhibition of phospholipase C activity significantly attenuates pressure-mediated afferent arteriolar vasoconstrictor responses. These observations are in good agreement with the findings of other investigators using arteries isolated from other vascular beds that exhibit myogenic or autoregulatory behavior well as with in vivo studies involving whole kidney hemodynamics. The results of this study support the hypothesis that calcium release from an IP3-sensitive intracellular calcium pool represents a critical link in the signal transduction pathway responsible for autoregulatory adjustments in afferent arteriolar diameter.
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Acknowledgments |
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These studies were supported by grants from the American Heart Association (95001370, 95009790) and the National Institutes of Health (DK 44628). Dr. Edward W. Inscho is an Established Investigator of the American Heart Association. Dr. John D. Imig is the recipient of a Grant-in-Aid from the American Heart Association.
Received September 16, 1997; first decision October 17, 1997; accepted October 29, 1997.
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References |
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1. Takenaka T, Harrison-Bernard LM, Inscho EW, Carmines PK, Navar LG. Autoregulation of afferent arteriolar blood flow in juxtamedullary nephrons. Am J Physiol. 1995; 267 : F879 –F887.
2. Arendshorst WJ, Navar LG. Renal circulation and glomerular hemodynamics. In: Schrier RW, Gottschalk C, eds. Diseases of the Kidney, 5th ed. Boston: Little, Brown & Co; 1993: 65 –117.
3. Ito S, Juncos LA, Carretaro OA. Pressure-induced constriction of the afferent arteriole of spontaneously hypertensive rats. Hypertension. 1992; 19 : II-164 –II-167.[Medline] [Order article via Infotrieve]
4. Holstein-Rathlou N, Marsh DJ. Renal blood flow regulation and arterial pressure fluctuations: a case study in nonlinear dynamics.
Physiol Rev. 1994;
74
: 637
–681.
5. Casellas D, Moore LC. Autoregulation of intravascular pressure in preglomerular juxtamedullary vessels. Am J Physiol. 1993; 264 : F315 –F321.[Medline] [Order article via Infotrieve]
6. Carmines PK, Mitchell KD, Navar LG. Effects of calcium antagonists on renal hemodynamics and glomerular function. Kidney Int. 1992; 41 :(suppl 36): S-43 –S-48.
7. Moore LC, Rich A, Casellas D. Ascending myogenic autoregulation: interactions between tubuloglomerular feedback and myogenic mechanisms. Bull Math Biol. 1994; 56 : 391 –410.[Medline] [Order article via Infotrieve]
8. Sanchez-Ferrer CF, Roman RJ, Harder DR. Pressure-dependent contraction of rat juxtamedullary afferent arterioles.
Circ Res. 1989;
64
: 790
–798.
9. Navar LG, Inscho EW, Majid DSA, Imig JD, Harrison-Bernard LM, Mitchell KD. Paracrine regulation of the renal microcirculation.
Physiol Rev. 1996;
76
: 425
–536.
10. Inscho EW, Cook AK, Navar LG. Pressure-mediated vasoconstriction of juxtamedullary afferent arterioles involves P2-purinoceptor activation. Am J Physiol. 1996; 271 : F1077 –F1085.[Medline] [Order article via Infotrieve]
11. Hayashi K, Epstein M, Loutzenhiser R. Pressure-induced vasoconstriction of renal microvessels in normotensive and hypertensive rats: Studies in the isolated perfused hydronephrotic kidney.
Circ Res. 1989;
65
: 1475
–1484.
12. Yip KP, Marsh DJ. [Ca2+], in rat afferent arteriole during constriction measured with confocal fluorescence microscopy. Am J Physiol. 1996; 271 : F1004 –F1011.[Medline] [Order article via Infotrieve]
13. Steinhausen M, Blum M, Fleming JT, Holz FG, Parekh N, Wiegman DL. Visualization of renal autoregulation in the split hydronephrotic kidney of rats. Kidney Int. 1989; 35 : 1151 –1160.[Medline] [Order article via Infotrieve]
14. Hayashi K, Epstein M, Saruta T. Altered myogenic responsiveness of the renal microvasculature in experimental hypertension. J Hypertens. 1996; 14 : 1387 –1401.[Medline] [Order article via Infotrieve]
15. Loutzenhiser R, Epstein M. Renal microvascular actions of calcium antagonists. J Am Soc Nephrol. 1990; 1 : S3 –S12.[Medline] [Order article via Infotrieve]
16. Casellas D, Moore LC. Autoregulation and tubuloglomerular feedback in juxtamedullary glomerular arterioles. Am J Physiol. 1990; 258 : F660 –F669.[Medline] [Order article via Infotrieve]
17. Conger JD, Falk SA, Robinette JB. Angiotensin II-induced changes in smooth muscle calcium in rat renal arterioles. J Am Soc Nephrol. 1993; 3 : 1792 –1803.[Abstract]
18. Mitchell KD, Navar LG. Tubuloglomerular feedback responses during peritubular infusions of calcium channel blockers. Am J Physiol. 1990; 258 : F537 –F544.[Medline] [Order article via Infotrieve]
19. Casellas D, Bouriquet N. Nimodipine-resistant tone in myogenically active preglomerular arteries of rat kidneys. Acta Physiol Scand. 1994; 152 : 345 –347.[Medline] [Order article via Infotrieve]
20. Casellas D, Carmines PK. Control of the renal microcirculation: Cellular and integrative perspectives. Curr Opin Nephrol Hypertens. 1996; 5 : 57 –63.[Medline] [Order article via Infotrieve]
21. Nelson MT, Conway MA, Knot HJ, Brayden JE. Chloride channels blockers inhibit myogenic tone in rat cerebral arteries.
J Physiol (Lond). 1997;
502
: 259
–264.
22. Ogawa N, Ono H. Effect of 8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release, on autoregulation of renal blood flow in the dog. Naunyn-Schmiedeberg’s Arch Pharmacol. 1988; 338 : 293 –296.[Medline] [Order article via Infotrieve]
23. Osol G, Laher I, Kelly M. Myogenic tone is coupled to phospholipase C and G protein activation in small cerebral arteries. Am J Physiol. 1993; 265 : H415 –H420.[Medline] [Order article via Infotrieve]
24. Harder DR, Gilbert R, Lombard JH. Vascular muscle cell depolarization and activation in renal arteries on elevation of transmural pressure. Am J Physiol. 1987; 253 : F778 –F781.[Medline] [Order article via Infotrieve]
25. Hill MA, Meininger GA. Calcium entry and myogenic phenomena in skeletal muscle arterioles. Am J Physiol. 1994; 267 : H1085 –H1092.[Medline] [Order article via Infotrieve]
26. Davis MJ, Donovitz JA, Hood JD. Stretch-activated single channel and whole cell currents in vascular smooth muscle cells. Am J Physiol. 1992; 262 : C1083 –C1088.[Medline] [Order article via Infotrieve]
27. Davis MJ, Meininger GA, Zaweja DC. Stretch-induced increases in intracellular calcium of isolated vascular smooth muscle cells. Am J Physiol. 1992; 263 : H1292 –H1299.[Medline] [Order article via Infotrieve]
28. Harder DR. Pressure-dependent membrane depolarization in cat middle cerebral artery.
Circ Res. 1984;
55
: 197
–202.
29. D’Angelo G, Meininger GA. Transduction mechanisms involved in the regulation of myogenic activity.
Hypertension. 1994;
23
: 1096
–1105.
30. McCarron JG, Crichton CA, Langton PD, MacKenzie A, Smith GL. Myogenic contraction by modulation of voltage-dependent calcium currents in isolated rat cerebral arteries.
J Physiol (Lond). 1997;
498
: 371
–379.
31. Karibe A, Watanabe J, Horiguchi S, Takeuchi M, Suzuki S, Funakoshi M, Katoh H, Keitoku M, Satoh S, Shirato K. Role of cytosolic Ca2+ and protein kinase C in developing myogenic contraction in isolated rat small arteries. Am J Physiol. 1997; 272 : H1165 –H1172.[Medline] [Order article via Infotrieve]
32. Scholz H, Kurtz A. Role of protein kinase C in renal vasoconstriction caused by angiotensin II. Am J Physiol. 1990; 259 : C421 –C426.[Medline] [Order article via Infotrieve]
33. Narayanan J, Imig M, Roman RJ, Harder DR. Pressurization of isolated renal arteries increases inositol trisphosphate and diacylglycerol. Am J Physiol. 1994; 266 : H1840 –H1845.[Medline] [Order article via Infotrieve]
34. Nakayama K. Calcium-dependent contractile activation of cerebral artery produced by quick stretch. Am J Physiol. 1982; 242 : H760 –H768.[Medline] [Order article via Infotrieve]
35. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci U S A. 1996; 87 : 2466 –2470.
36. Lytton J, Westlin M, Hanley MR. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.
J Biol Chem. 1991;
266
: 17067
–17071.
37. Mason MJ, Garcia-Rodriguez C, Grinstein S. Coupling between intracellular Ca2+ stores and the Ca2+ permeability of the plasma membrane.
J Biol Chem. 1991;
266
: 20856
–20862.
38. Seidler NW, Jona I, Vegh M, Martonosi A. Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum.
J Biol Chem. 1989;
264
: 17816
–17823.
39. Uyama Y, Imaizumi Y, Watanabe M. Cyclopiazonic acid, an inhibitor of Ca2+-ATPase in sarcoplasmic reticulum, increases excitability in ileal smooth muscle. Br J Pharmacol. 1993; 110 : 565 –572.[Medline] [Order article via Infotrieve]
40. Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE. Receptor-coupled signal transduction in human neutrophils: Effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness.
J Pharmacol Exp Ther. 1990;
253
: 688
–697.
41. Casellas D, Navar LG. In vitro perfusion of juxtamedullary nephrons in rats. Am J Physiol. 1984; 246 : F349 –F358.[Medline] [Order article via Infotrieve]
42. Inscho EW, Imig JD, Cook AK. Afferent and efferent arteriolar vasoconstriction to angiotensin II and norepinephrine involves release of Ca2+ from intracellular stores.
Hypertension. 1997;
29
: 222
–227.
43. Grover AK, Xu A, Samson SE, Narayanan N. Sarcoplamic reticulum Ca2+ pump in pig coronary artery smooth muscle is regulated by a novel pathway. Am J Physiol. 1996; 271 : C181 –C187.[Medline] [Order article via Infotrieve]
44. Goeger DE, Riley RT, Dorner JW, Cole RJ. Cyclopiazonic acid inhibition of the Ca2+-transport ATPase in rat skeletal muscle sarcoplasmic reticulum vesicles. Biochem Pharmacol. 1988; 37 : 978 –981.[Medline] [Order article via Infotrieve]
45. Inscho EW, Ohishi K, Cook AK, Belott TP, Navar LG. Calcium activation mechanisms in the renal microvascular response to extracellular ATP. Am J Physiol. 1995; 268 : F876 –F884.[Medline] [Order article via Infotrieve]
46. Bell PD. Calcium antagonists and intrarenal regulation of glomerular filtration rate. Am J Nephrol. 1987; 7 : 24 –31.[Medline] [Order article via Infotrieve]
47. Bell PD, Reddington M. Intracellular calcium in the transmission of tubuloglomerular feedback signals. Am J Physiol. 1983; 245 : F295 –F302.[Medline] [Order article via Infotrieve]
48. Yanagisawa T, Okada Y. KCl depolarization increases Ca2+ sensitivity of contractile elements in coronary arterial smooth muscle. Am J Physiol. 1994; 267 : H614 –H621.[Medline] [Order article via Infotrieve]
49. Suzuki M, Muraki K, Imaizumi Y, Watanabe M. Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+-pump, reduces K+ currents in guinea-pig smooth muscle cells. Br J Pharmacol. 1992; 107 : 134 –140.[Medline] [Order article via Infotrieve]
50. Omote M, Mizusawa H. Rhythmic relaxations of active tension in the rabbit large arteries induced by a combination of cyclopiazonic acid and Bay K 8644. Br J Pharmacol. 1996; 118 : 233 –236.[Medline] [Order article via Infotrieve]
51. Xuan Y, Wang O, Whorton R. Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways. Am J Physiol. 1992; 262 : C1258 –C1265.[Medline] [Order article via Infotrieve]
52. Buryi V, Morel N, Salomone S, Kerger S, Godfraind T. Evidence for a direct interaction of thapsigargin with voltage-dependent Ca2+ channel. Naunyn-Schmiedeberg’s Arch Pharmacol. 1995; 351 : 40 –45.[Medline] [Order article via Infotrieve]
53. Rossier MF, Python CP, Burnay MM, Schlegel W, Vallotton MB, Capponi AM. Thapsigargin inhibits voltage activated calcium channels in adrenal glomerulosa cells. Biochem J. 1993; 296 : 309 –312.[Medline] [Order article via Infotrieve]
54. Sekiguchi F, Shimamura K, Akashi M, Sunano S. Effects of cyclopiazonic acid and thapsigargin on electromechanical activities and intracellular Ca2+ in smooth muscle of carotid artery of hypertensive rats. Br J Phannacol. 1996; 118 : 857 –864.
55. Imig JD, Cook AK, Inscho EW. Postglomerular vasoconstriction to angiotensin and norepinephrine depend on release of Ca2+ from intracellular stores. Microcirculation. 1997; 4 : 182 . Abstract.
56. Berridge MJ. Inositol trisphosphate and calcium signaling. Nature. 1993; 361 : 315 –325.[Medline] [Order article via Infotrieve]
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