AJP - Regu Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 293: R2225-R2231, 2007. First published October 10, 2007; doi:10.1152/ajpregu.00287.2007
0363-6119/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/R2225    most recent
00287.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hultström, M.
Right arrow Articles by Persson, A. E. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hultström, M.
Right arrow Articles by Persson, A. E. G.

RENAL HEMODYNAMICS AND CARDIORENAL INTEGRATION

Adenosine triphosphate increases the reactivity of the afferent arteriole to low concentrations of norepinephrine

Michael Hultström,1,* En Yin Lai,1,* Zufu Ma,1 Örjan Källskog,1 Andreas Patzak,2 and A. Erik G. Persson1

1Department of Medical Cell Biology, Division of Physiology, University of Uppsala, Uppsala, Sweden, 2Johannes-Müller-Institute of Physiology, University Hospital Charité, Humboldt-University of Berlin

Submitted 26 April 2007 ; accepted in final form 6 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosine triphosphate (ATP) and norepinephrine (NE) interact in the control of blood flow in the kidney. A combined effect of NE and ATP has not been previously investigated at the level of the afferent arteriole (Af). We studied the effects of ATP on the contractile response of the Af to NE. Vascular reactivity to ATP, NE, and their combination was investigated in isolated perfused Af from mice. The roles of {alpha}-adrenoceptors and P2-ATP-receptors were investigated by use of specific agonists and antagonists. Cytosolic calcium was measured using the fluorescent calcium dye fura-2. ATP in concentrations from 10–12 to 10–4 mol/l induced transient contractions. NE constricted the Af in a dose-dependent manner and induced significant contractions at > 10–7 mol/l. Treatment with ATP (10–8 and 10–6 mol/l) increased the NE response. Diameters were reduced by 20% already at 10–11 mol/l NE during ATP treatment of 10–6 mol/l. ATP increased the calcium response to NE significantly at 10–8 and 10–7mol/l NE. The P2-type ATP receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10–5 mol/l) abolished the sensitization of the NE response by ATP. The {alpha}1-blocker prazosin (10–7 mol/l) inhibited the ATP effect, as did the {alpha}2-blocker yohimbine (10–7 mol/l). Neither the phenylephrine- nor clonidine-induced concentration response curves was affected by ATP in the bath solution. Costimulation with ATP enhances the response of the Af to NE. This effect is mediated by increased cytosolic calcium. The enhancing effect involves P2-type ATP receptors and both {alpha}1- and {alpha}2-adrenoceptors.

cytosolic calcium; mice; prazosin; PPADS; renal circulation; yohimbine

AFFERENT ARTERIOLES ARE PIVOTAL to the regulation of renal blood flow and glomerular filtration rate because of their significant contribution to total renal resistance and their key position in control systems. The tone of afferent arterioles is determined by the action of the sympathetic nervous system and several endocrine and paracrine factors (16) as well as by autoregulation (1). NE is the primary peripheral neurotransmitter of the sympathetic nervous system. In the kidney, norepinephrine (NE) has been shown to be important for the regulation of the afferent arteriolar tone through its {alpha}1-receptor (5). Activation of {alpha}2-receptor has little effect in itself but has been shown to augment the response to ANG II (18). Adrenergic β-receptors are of lesser importance in the renal vasculature (4). There are two cotransmitters of NE, ATP, and neuropeptide-Y that contribute to the effect of sympathetic nerves on renal vessels (25). ATP itself affects renal vessels acting on P2X-receptors causing the afferent arterioles to constrict (14–16). Recent results indicate that the interaction of ATP and NE may be important in the regulation of vascular tone at the afferent arteriole and its regulation of renal function. In the isolated perfused kidney, inhibition of ATP P2-receptors decrease the pressor response of renal nerve stimulation (34). ATP has also been shown to modulate the different vascular responses to sympathetic nerve stimulation and to exogenous NE in the kidneys (31). In addition to its corelease from sympathetic nerves, ATP may be released from macula densa cells (3, 19) and play a role in mediating the tubuloglomerular feedback (TGF) in the kidney (16, 28). However, the effect of combined ATP and NE stimulation has not been investigated at the level of the afferent arteriole.

The aim of the present study was to investigate how ATP interacts with NE in controlling the afferent arteriolar tone and to study which receptors are involved in regulating such an effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male mice of the C57 black 6J strain (Scanbur BK, Solna, Sweden) were used in these experiments. The mice weighed between 25 and 30 g. Animals were fed standard pelleted food (Scanbur BK) and had free access to tap water. The experiments were ethically approved by the board of animal experiments at the county court of Uppsala.

Dissection and perfusion. Dissection and perfusion were performed as previously published (26). In short, the animals were killed by cervical dislocation, and the kidneys were removed and immediately placed in ice-cold DMEM with 0.1% albumin. The kidneys were sliced in thin transversal sections, and the afferent arterioles were dissected by using sharpened forceps (no. 5; Dumont) under a stereomicroscope (Leica Microsystems, Wetzlar, Germany).

Arterioles from the outer cortex with attached glomerulus were selected and transferred to a thermoregulated chamber on the stage of an inverted microscope (Noran, Middleton, WI). Glass pipettes (Drummond Scientific, Broomall, PA) mounted in a perfusion system (Vestavia Scientific, Vestavia Hills, AL) were used to fix and perfuse the arteriole and glomerulus (Fig. 1A). The holding pipette (OD, 2.13 mm; ID, 1.63 mm) had an aperture of roughly 26 µm at the tip and a constriction of about 20 µm after customizing. The proximal end of the arteriole was aspirated into this pipette. The inner, perfusion pipette (OD, 1.19 mm; ID, 1.02 mm) with a diameter of the tip of 5 µm was advanced into the lumen of the arteriole. This pipette was connected to a reservoir containing the perfusion solution and to a manometer. The arterioles were perfused at 37°C with a pressure of 100 mmHg in the pressure head, which we have previously shown produces a perfusion pressure within the autoregulatory range and with a physiological perfusion flow rate (26). Arterioles that exhibited a basal tone and clear lumen (Fig. 1B) were tested for viability by exchanging the bath solution to a 100 mmol/l KCl after which the arterioles were allowed to recover for 10 min. Only arterioles showing a strong contraction to the KCl challenge, as shown in Fig. 1C, were used.


Figure 1
View larger version (66K):
[in this window]
[in a new window]

  		Fig. 1. Perfusion setup as seen under the microscope. A: shows the holding pipette (ghp) to the left holding the glomerulus (g) and the perfusion pipette to the right with an outer pipette (ahp) for holding the afferent arteriole and an inner pipette (pp) introduced into the afferent arteriole (af) for perfusing the same. A tubulus fragment (t) is attached to the glomerulus. B and C: the arteriole at experimental magnification, first relaxed with a clear lumen (B) and then fully contracted (C). Scale bars = 10 µm.

 
Measurement of arteriolar diameter. These experiments were recorded on super video home system videotapes (video recorder Panasonic NV-HS830; Matsushita Audio Video, Lueneburg, Germany). The end-magnification results were from a Nikon x60/1.2 water-immersion objective lens and projection (x1) on a 0.3'' chip digital camera (model VCAM110; Phytec Technology Holding, Mainz, Germany). Video sequences were digitized by using a frame-grabber card (pciGrabber-4plus; Phytec Technology Holding). The vessel diameters were determined by using customized software (from Dr. H. Siegmund, Johannes-Müller-Institute of Physiology, Humboldt-University of Berlin, Germany). The equipment allowed a resolution of 0.11 µm of the vessel structure. The luminal diameter of the afferent arteriole was measured at one point in five consecutive pictures with 1-s intervals. Each vessel was used for one experiment only. Control diameter was obtained after the recovery from the KCl test, shortly before starting the experimental protocol.

Measurement of intracellular calcium. In separate experiments, afferent arterioles were dissected and perfused as described above and incubated with fura-2 AM (VWR International, Stockholm, Sweden) at 10–5 mol/l for 45 min. The arterioles were excited alternately at 340 and 380 nm and the emission was measured at 510 nm with 3-s collection periods for each excitation. The ratio between emissions in successive time periods (340/380 nm) was used to determine intracellular calcium concentration against a calibrated standard (model F-6774 fura-2 calcium imaging calibration kit; Molecular Probes) in the same microscope. Fluorescence was detected using an Applied Imaging Quanticell-900 (VisiTech International, Sunderland, UK). Three regions of interest were selected on each vessel, one covering the whole width of the vessel and one from each side of the vessel wall as shown in Fig. 2A. The tracings from these areas generally were very similar. Results from the three areas were averaged for each vessel. The fura-2 ratio was measured for 2 min after application of each incremental dose of NE. The calcium value for the baseline was acquired before application of agonist, the peak value was selected manually, and the plateau value was acquired from the collection periods ~105 s after the application of agonist, corresponding to the measurement time for the sustained contraction.


Figure 2
View larger version (55K):
[in this window]
[in a new window]

  		Fig. 2. Example pictures from a calcium experiment. A: placement of the areas of interest and glomerulus (Glom.) indicated. B, C, and D: show fluorescence from excitation at 340 nm at the time of the baseline (B), peak (C), and plateau (D) while E, F, and G show fluorescence from excitation at 380 nm for the same time points.

 
Diameter time-response curves. The time response of the afferent arterioles was investigated for ATP at a concentration of 10–6 mol/l and NE at 10–4 mol/l. Each experiment was 3 min long, and measurements were taken every 6 s. After 1 min, the bath solution was exchanged to a solution with the above-indicated concentrations of ATP or of NE. The curves were adjusted so that the first strong contraction after superfusion with agonist was superimposed for the individual curves, thereby giving a more accurate time illustration of the time response (Figs. 3A and 5A ).


Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 3. A: diameter time-response curve with application of 10–6 mol/l ATP. Solid triangles indicate the mean and the error bars are SE (n = 7). B: example tracing of the intracellular calcium transient elicited by 10–6 mol/l ATP. Scale bar = 100 nmol/l. C: averaged calcium transients elicited by 10–6 mol/l ATP. *Significant increase compared with the baseline; n = 7.

 

Figure 5
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 5. A: diameter time-response curve with application of 10–4 mol/l NE. Mean {blacksquare} and the error bars (SE); n = 5. B: example tracing of the intracellular calcium transient elicited by 10–4 mol/l NE. Scale bar = 100 nmol/l. C: averaged calcium transients elicited by 10–4 mol/l NE. *Significant increase compared with the baseline; n = 6.

 
Diameter dose-response curves. A normal dose-response curve for NE was obtained by exchanging the bath solution at 2-min intervals in steps of 10x dilution from 10–12 to 10–4 mol/l. The vessel diameter was measured at the end of each period as a measure of sustained vessel diameter. The ATP dose-response curve was obtained in a similar manner.

ATP effect on NE diameter dose response. To study the effect of ATP on the dose-response curve for NE, the bath solution was exchanged to solutions containing different concentrations of ATP (10–10, 10–8, and 10–6 mol/l). Thereafter, the ATP concentration was kept constant during the measurement of the dose-response reaction of the afferent arteriole to NE from 10–12 to 10–4 mol/l.

Use of inhibitors. All antagonists were bought from Sigma (St. Louis, MO). When the effects of specific receptor blockers on the interaction between NE and ATP were investigated, the receptor antagonists were added together with ATP and a NE dose-response curve was recorded as described above with constant concentrations of ATP and the antagonist. The receptor antagonists used were the {alpha}1-adrenoceptor blocker prazosin at a concentration of 10–7 mol/l, the {alpha}2-adrenoceptor blocker yohimbine at a concentration of 10–7 mol/l, and the ATP-receptor blocker PPADS at a concentration of 10–5 mol/l as has been previously shown to be sufficient in vascular preparations from the mouse (10, 27).

Diameter dose-response curves for specific agonists. To test whether either {alpha}1- or {alpha}2-adrenoceptor activation is sufficient for the effects of ATP on the NE dose response, we recorded additional dose-response curves for the {alpha}1-agonist phenylephrine and the {alpha}2-agonist clonidine (both from Sigma) with and without ATP in the bath solution as described for NE above.

Statistics. The curves were analyzed with two-way ANOVA to test for contraction over time and difference between curves. Tukey's honestly significant difference test was used as post hoc test between curves, and paired t-tests were used to test for significant calcium increase compared with baseline. P < 0.05 was considered significant. Statistics were calculated using R 2.1.0 (32).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ATP elicited contraction and calcium transients. Bolus application of 10–6 mol/l ATP caused a strong (77 ± 13%, P < 0.05) but transient contraction in the afferent arteriole. The maximum contraction was reached during the first measurement interval after superfusion with agonist. The arterioles reopen to the control diameter after the bolus application (Fig. 3A). The transient contraction was accompanied by a small (from 185 ± 7 nM to 227 ± 11.6 nM, P < 0.05) increase in intracellular calcium exhibiting a flat time course without clear peak or any sustained response (exemplified in Fig. 3B with averages in 3C).

NE-elicited contraction and calcium transients. NE (10–4 mol/l) elicited a complete and stable contraction in all arterioles. The arterioles contracted within one to two measurement intervals (6–12 s) by 99 ± 1% (P < 0.05) and remained fully contracted during the whole time of measurement (Fig. 5A). The calcium response to 10–4 mol/l NE showed a peak increase from 171 ± 24 nM to 334 ± 83 nM (P < 0.05) and a sustained plateau at 226 ± 29 nM (P < 0.05) for the full 2 min of measurement as shown in Fig. 5, B and C (Fig. 2, B–G, shows corresponding original fluorescence images).

Sustained contractions elicited by ATP and NE. The sustained response to successive applications of ATP from 10–12 to 10–4 mol/l showed no diameter change compared with the baseline at any concentration (Fig. 4, A and B). NE alone elicited no transient or sustained vascular reaction below 10–7 mol/l in any vessel and showed a 44 ± 14% contraction around 10–6 mol/l from a basal diameter of 10 ± 0.9 µm. At 10–5 and 10–4 mol/l, NE elicited a near-complete contraction of the afferent arteriole (95 ± 2% and 96 ± 2%, respectively) (Fig. 6, {blacksquare}).


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. A: effect on afferent arteriolar diameter of increasing concentrations of ATP ({blacksquare}). Diameter was measured 105 s after application of the drug. (n = 5). B: same dose-response curves as shown in A expressed as %baseline diameter.

 

Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 6. A: dose-response curves for NE with different concentrations of ATP in the bath solution. Dose response of NE alone ({blacksquare}; n = 6); effect of 10–8 mol/l ATP on the NE dose-response (bullet; *significant difference compared with NE alone; n = 5); effect of 10–6 mol/l ATP on the NE dose-response ({blacktriangleup}; {dagger}significant difference from NE alone; n = 8). B: Same dose-response curves as shown in A are expressed as %baseline diameter. Dose response of NE alone {blacksquare}; effect of 10–8 mol/l ATP on the NE dose-response bullet observed to elicit contraction at a significantly lower dose (*significant difference compared with NE alone); effect of 10–6 mol/l ATP on the NE dose-response ({blacktriangleup}; {dagger}significant difference from NE alone).

 
ATP effect on NE-elicited contraction. The combination of ATP and NE partially increased the sensitivity of the afferent arteriole to NE. ATP concentrations of 10–8 and 10–6 mol/l in bath solution caused a reaction to NE at successively lower concentrations (Fig. 6, A and B). ATP (10–10 mol/l) did not affect the NE dose-response curve (P > 0.2 by two-way ANOVA) having a basal diameter of 9.1 ± 0.6 µm and showing a 64 ± 21% contraction at 10–6 mol/l NE and 98 ± 1% and 96 ± 2% at 10–4 and 10–5 mol/l NE. The contraction caused by 10–6 mol/l and 10–8 mol/l ATP in combination with NE was significant already at 10–11 mol/l NE (21 ± 5%) and 10–8 mol/l NE (22 ± 6%), respectively, with baselines at 6.3 ± 0.9 µm and 8.4 ± 0.7 µm. The dose-response curves to NE in combination with 10–8 mol/l and 10–6 mol/l ATP show a bimodal course. The sensitized contraction at low concentrations decreased the resting diameter of the afferent arteriole with ~20%. A second phase of contraction was evident at 10–6 mol/l NE and above. This resulted in a 92 ± 5% at 10–4 mol/l NE in both groups (Fig. 6, A and B).

ATP effect on NE elicited calcium transients. Cytosolic calcium transients elicited by successive applications of NE between 10–12 mol/l and 10–4 mol/l showed no change at concentrations lower than 10–7 mol/l. At 10–7 mol/l NE a small calcium increase (from 168 ± 20 nM to 191 ± 22 nM) was detectable but without sustained calcium increase. Higher concentrations of NE induced a strong calcium increase with a clear peak/plateau time course with a calcium increase at 10–6 mol/l NE of 93 ± 27 nM or 52% and a plateau phase at 58 ± 20 nM above the baseline at 177 nM (Fig. 7A). With 10–6 mol/l ATP in the bath solution the calcium increase is stronger than for NE alone (from 168 ± 9 nM to 192 ± 8 nM at 10–8 mol/l NE and from 166 ± 4 nM to 204 ± 6 nM at 10–7 mol/l NE (Fig. 7B, P < 0.05 for both). The response at 10–6 mol/l NE is similar to that of NE alone (Fig. 7B).


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 7. A: dose-response curve, showing averaged calcium transients from afferent arterioles stimulated with NE as bars ({ddagger}significant increase in intracellular calcium compared with the baseline for the same concentration). The change above the baseline of the peak response is depicted (bullet). For this line the asterisk denotes significant difference compared with the curve for NE with 10–6 mol/l ATP shown in B by 2-way ANOVA. {dagger}Significant difference in peak response compared with the curve for NE with 10–6 mol/l ATP shown in B (n = 5). B: dose-response curve showing averaged calcium transients from afferent arterioles stimulated with NE and 10–6 mol/l ATP in combination, shown as bars ({ddagger}significant increase in intracellular calcium compared with the baseline for the same concentration). The change above the baseline of the peak response is depicted as filled circles (*significant difference compared with the curve for NE alone, shown in A by 2-way ANOVA). {dagger}Significant difference in peak response compared with the curve for NE alone, shown in A (n = 6).

 
Receptor antagonists. Blocking the P2-type ATP receptors with 10–5 mol/l PPADS during performance of a NE dose-response curve with 10–6 mol/l ATP in the bath solution gives a curve that is similar to that of NE alone (Fig. 8, A and B) and significantly different from the NE dose-response curve with 10–6 mol/l ATP (P < 0.05 by two-way ANOVA). The {alpha}1-adrenoceptor blocker prazosin completely blocked the effect of ATP at low concentrations of NE and shifted the contraction of the afferent arteriole to the right (P < 0.05 by two-way ANOVA) (Fig. 8, C and D). The baseline diameter of the prazosin group was larger than that of the group treated with NE and 10–6 mol/l ATP. However, when comparing the baseline diameters of all groups, there are no significant differences between groups and we believe the differences shown are illustrative of the variation in basal afferent arteriolar diameters. The {alpha}2-adrenoceptor yohimbine was also able to block the ATP effect (P < 0.05 compared with NE with 10–6 mol/l ATP) without changing the contraction caused by NE (P > 0.6 compared with NE alone by two-way ANOVA) (Fig. 8, E and F).


Figure 8
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 8. A: ATP P2 receptor blocker PPADS inhibits the effect of 10–6 mol/l ATP on the NE dose-response curve (n = 6). B: data in A as %contraction (*significant difference from NE with 10–6 mol/l ATP, {blacktriangleup} in Fig. 6). The curve is similar to the normal NE dose-response curve (Fig. 6, {blacksquare}) and different from the NE + 10–6 mol/l ATP curve (Fig 6, {blacktriangleup}) by two-way ANOVA. C: prazosin ({alpha}1-adrenoceptor blocker) inhibits the effect of 10–6 mol/l ATP on the NE dose-response curve (n = 7, *significant difference from NE with 10–6 mol/l ATP, {blacktriangleup} in Fig. 6). The curve is different from both the normal NE dose-response curve (Fig. 6, {blacksquare}) and NE + 10–6 mol/l ATP (Fig 6, {blacktriangleup}) by two-way ANOVA (P < 0.05). The baseline diameter is larger than that of the NE + 10–6 mol/l ATP group (Fig 6, {blacktriangleup}) when analyzed in isolation, but there is no difference between the baseline diameters of all groups by ANOVA with Tukey's post hoc test. D: graph showing the data in C as %contraction (*significant difference from NE with 10–6 mol/l ATP, {blacktriangleup} in Fig. 6). The curve is different from both the normal NE dose-response curve (Fig. 6, {blacksquare}) and NE + 10–6 mol/l ATP (Fig 6, {blacktriangleup}) by 2-way ANOVA (P < 0.05). E: yohimbine ({alpha}2-adrenoceptor blocker) inhibits the effect of 10–6 mol/l ATP on the NE dose-response curve (n = 5, *significant difference from NE with 10–6 mol/l ATP, {blacktriangleup} in Fig. 6). The curve is similar to the normal NE dose-response curve (Fig. 6, {blacksquare}) and different from the NE + 10–6 mol/l ATP curve (Fig 6, {blacktriangleup}) by 2-way ANOVA (P < 0.05). F: data in E as %contraction (*significant difference from NE with 10–6 mol/l ATP, {blacktriangleup} in Fig. 6). The curve is similar to the normal NE dose-response curve (Fig. 6, {blacksquare}) and different from the NE + 10–6 mol/l ATP curve (Fig 6, {blacktriangleup}) by 2-way ANOVA (P < 0.05).

 
Specific {alpha}-adrenergic agonists. The {alpha}1-agonist phenylephrine elicited a reaction similar to the endogenous agonist NE (P > 0.5 by ANOVA, n = 6) showing a 31 ± 8.5% contraction at 10–6 mol/l and a near-full contraction at higher concentrations (70 ± 2.7% and 85 ± 2.2% from a 11.0 ± 1.1 µm baseline). However, the dose-response curve of phenylephrine showed no change under the influence of 10–6 mol/l ATP (P > 0.4 by ANOVA, n = 5) with a 43 ± 13% contraction at 10–6 mol/l and 82 ± 6% and 90 ± 3% at the higher concentrations with the baseline at 9.8 ± 0.5 µm.

In response to the {alpha}2-agonist clonidine the afferent arterioles contracted at 10–5 and 10–4 mol/l (14 ± 5% and 18 ± 5% from a 8.4 ± 1.1-µm baseline, respectively; P < 0.05; n = 6). No further contraction was seen when clonidine was combined with ATP (P > 0.4 by two-way ANOVA, n = 6) showing a 11 ± 4% contraction at 10–5mol/l and 12 ± 4% at 10–4 mol/l from the 10.0 ± 0.6-µm baseline (P < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main finding presented in this paper is that ATP increases the response of the afferent arteriole to low concentrations of NE through a pathway depending on P2-type ATP receptors and both the {alpha}1- and {alpha}2-type adrenoceptors.

The finding of a strong contractile response to 10–6 mol/l ATP (Fig. 3A) is consistent with earlier findings (16). On the other hand, we have not been able to show any sustained contraction to increasing concentrations of ATP. This is in contrast to work by Inscho et al. (16) and others (36) where ATP is shown to elicit a sustained contraction on the afferent arteriole in the juxtamedullary nephron preparation at 10–6 mol/l, causing about 17% constriction. That we do not see this contraction in the isolated afferent arteriole may reflect differences both in species and in the preparations. While we see a small calcium increase after application of 10–6 ATP (Fig. 3B), it is not sufficient to cause a sustained contraction (Fig. 3A). These results indicate that the afferent arteriole from the mouse may have a different pharmacological profile than that from the rat. Both P2X and P2Y receptors have been found in the intrarenal arteries, and both receptor types are involved in regulating the pressor response to renal nerve stimulation (34). In the rat, ATP exerts its paracrine or neurotransmitter function through P2X receptors on the interlobular arteries and afferent arterioles (15, 36). In contrast, in the rabbit, P2X inhibition has been shown not to effect the renal blood flow response to renal nerve stimulation (9).

The 10–4 mol/l NE produced a quickly established, stable contraction (Fig. 5A), which is in agreement with earlier results from our laboratory showing no weakening of calcium transients with successive applications of NE (20). In addition, it is in agreement with the earliest results in the isolated perfused afferent arteriole of rabbits showing sustained contraction over a 3-min measurement period (6) and with studies in mouse arterioles (13). The response has slightly lower sensitivity than that seen in the rabbit (6, 7), while the rat exhibits a more gradual contraction from 10–9 to 10–5 mol/l (35). The strong calcium increase upon stimulation with 10–4 mol/l NE with a clear peak/plateau time course indicates that the response to NE is significantly mediated by changes in the cytosolic calcium. This observation is consistent with earlier findings (20, 21).

ATP concentrations of 10–8 and 10–6 mol/l sensitized the afferent arteriole to low concentrations of NE in a dose-dependent manner (Fig. 6). A sensitizing effect of ATP on the afferent arteriole to NE stimulation can also be concluded from results from renal nerve stimulation of the isolated perfused kidney during P2-receptor inhibition (34). This suggests an enhancing effect of ATP on NE-induced contractions of glomerular arterioles that with a 20% decrease in diameter would cause a 240% increase in resistance and may cause as much as a 60% decrease of renal blood flow if the average is extrapolated to all nephrons (29). This may provide a mechanism for the resetting of TGF, given that ATP is released from macula densa cells upon TGF activation (3) and the fact that sympathetic stimulation has been shown to reset TGF (12, 22). It may further indicate that ATP may act to enhance not only the effect of direct sympathetic activity by also adrenergic stimulation at extra junctional sites, which has been proposed as an important signaling pathway given that the range of physiological responses shown for sympathetic activity does not clearly coincides with anatomical findings (2).

Baseline calcium measurements are comparable to what we have previously shown to be normal for the distal afferent arteriole with attached glomerulus (11, 21). Although the dose response of calcium to increasing concentrations of NE was statistically increased by costimulation with 10–6 mol/l ATP at 10–8 and 10–7 mol/l NE, the numerical difference was small (Fig. 7, A and B) compared with the contractile response seen at lower concentrations (Fig. 5). This suggests that the increased sustained contraction is not only induced by calcium-dependent signaling, but may also depend on greater sensitivity of the contractile apparatus to calcium as it has been shown for the adenosine ANG II interaction (23). It has been shown that calcium sensitivity may increase through activation of Rho kinase, protein kinase C, and p38 mitogen activated protein kinase leading to increased phosphorylation of the myosin light chain (23, 30, 33).

Our results indicate that activation of a P2-type ATP receptor is necessary for the increased response to NE seen in the afferent arteriole of the mouse (Fig. 8A). Both the {alpha}2-blocker yohimbine and the {alpha}1-receptor blocker prazosin were able to block the sensitization (Fig. 8, B and C). This may indicate a general dependence on {alpha}-receptors. However, one has to take into consideration that prazosin is not completely selective in the mouse (17, 24).

The rightward shift of the dose-response curve of NE in response to prazosin (Fig. 8B) indicates a considerable role of the {alpha}1-receptor in mediating NE-induced contractions. This is consistent with findings by other investigators (4, 7). The minimal effect of {alpha}2-inhibition on the contraction caused by NE (Fig. 7C) suggests a minor contribution of {alpha}2-receptors to the contraction caused by NE alone. The finding is also in agreement with earlier results (4, 7). There are, however, results showing that both {alpha}1- and {alpha}2-receptors significantly contribute the effect of sympathetic stimulation on renal blood flow (8). This may be interpreted as consistent with the present finding of a dual dependence on {alpha}1- and {alpha}2-receptors for the effect of ATP costimulation on afferent arteriolar sensitivity to NE.

The idea that both {alpha}1- and {alpha}2-adrenoceptors are important for the interaction of ATP with NE is further supported by the inability of ATP to change the dose-response curves of the selective agonists phenylephrine and clonidine.

In conclusion, we have shown that the enhancing effect of ATP on sympathetic regulation of renal blood flow may be explained by mechanisms intrinsic to the afferent arteriole. Furthermore, the sensitized response to NE by costimulation with ATP is dependent on both {alpha}1- and {alpha}2-adrenoceptors and P2-type ATP receptors.

Perspectives and Significance

The present work may serve to highlight the importance of interactions between different transmission systems in the kidney. An observation of interest is the relatively small changes in calcium signaling seen during the augmented contractions, indicating a significant increase in calcium sensitivity or the activation of calcium-independent contraction. This is an area that warrants deeper investigation to completely understand the mechanisms. Furthermore, the interaction of ATP and NE signaling may provide a mechanism for the interaction between the TGF and sympathetic activity reported earlier.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Hultström, Renal Research Group, Dept. of Internal Medicine, Haukeland Univ. Hospital, Jonas Liesvei 65, 5021 Bergen, Norway (e-mail: michael.hultstrom{at}med.uib.no)

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.

* M Hultström and E. Y. Lai contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aukland K, Oien AH. Renal autoregulation: models combining tubuloglomerular feedback and myogenic response. Am J Physiol Renal Fluid Electrolyte Physiol 252: F768–F783, 1987.[Abstract/Free Full Text]
  2. Barajas L, Powers K. Monoaminergic innervation of the rat kidney: a quantitative study. Am J Physiol Renal Fluid Electrolyte Physiol 259: F503–F511, 1990.[Abstract/Free Full Text]
  3. Bell PD, Lapointe JY, Sabirov R, Hayashi S, Peti-Peterdi J, Manabe K, Kovacs G, Okada Y. Macula densa cell signaling involves ATP release through a maxi anion channel. Proc Natl Acad Sci USA 100: 4322–4327, 2003.[Abstract/Free Full Text]
  4. DiBona GF, Kopp UC. Neural control of renal function. Physiol Rev 77: 75–197, 1997.[Abstract/Free Full Text]
  5. DiBona GF, Sawin LL. Role of renal {alpha}2-adrenergic receptors in spontaneously hypertensive rats. Hypertension 9: 41–48, 1987.[Abstract/Free Full Text]
  6. Edwards RM. Segmental effects of norepinephrine and angiotensin II on isolated renal microvessels. Am J Physiol Renal Fluid Electrolyte Physiol 244: F526–F534, 1983.[Abstract/Free Full Text]
  7. Edwards RM, Trizna W. Characterization of {alpha}-adrenoceptors on isolated rabbit renal arterioles. Am J Physiol Renal Fluid Electrolyte Physiol 254: F178–F183, 1988.[Abstract/Free Full Text]
  8. Eppel GA, Lee LL, Evans RG. {alpha}-Adrenoceptor subtypes mediating regional kidney blood flow responses to renal nerve stimulation. Auton Neurosci 112: 15–24, 2004.[CrossRef][ISI][Medline]
  9. Eppel GA, Ventura S, Denton KM, Evans RG. Lack of contribution of P2X receptors to neurally mediated vasoconstriction in the rabbit kidney in vivo. Acta Physiol (Oxf) 186: 197–207, 2006.[CrossRef][Medline]
  10. Guns PJ, Korda A, Crauwels HM, Van Assche T, Robaye B, Boeynaems JM, Bult H. Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta. Br J Pharmacol 146: 288–295, 2005.[CrossRef][ISI][Medline]
  11. Gutierrez AM, Kornfeld M, Persson AE. Calcium response to adenosine and ATP in rabbit afferent arterioles. Acta Physiol Scand 166: 175–181, 1999.[CrossRef][ISI][Medline]
  12. Hermansson K, Kallskog O, Wolgast M. Effect of renal nerve stimulation on the activity of the tubuloglomerular feedback mechanism. Acta Physiol Scand 120: 381–385, 1984.[ISI][Medline]
  13. Imig JD, Breyer MD, Breyer RM. Contribution of prostaglandin EP2 receptors to renal microvascular reactivity in mice. Am J Physiol Renal Physiol 283: F415–F422, 2002.[Abstract/Free Full Text]
  14. Inscho EW, Cook AK. P2 receptor-mediated afferent arteriolar vasoconstriction during calcium blockade. Am J Physiol Renal Physiol 282: F245–F255, 2002.[Abstract/Free Full Text]
  15. Inscho EW, Cook AK, Imig JD, Vial C, Evans RJ. Physiological role for P2X1 receptors in renal microvascular autoregulatory behavior. J Clin Invest 112: 1895–1905, 2003.[CrossRef][ISI][Medline]
  16. Inscho EW, Mitchell KD, Navar LG. Extracellular ATP in the regulation of renal microvascular function. FASEB J 8: 319–328, 1994.[Abstract]
  17. Ireland LM, Cain JC, Rotert G, Thomas S, Shoghi S, Hancock AA, Kerwin JF Jr. [3H]R-terazosin binds selectively to {alpha}1-adrenoceptors over {alpha}2-adrenoceptors–comparison with racemic [3H]terazosin and [3H]prazosin. Eur J Pharmacol 327: 79–86, 1997.[CrossRef][ISI][Medline]
  18. Jackson EK, Herzer WA, Kost CK Jr, Vyas SJ. Enhanced interaction between renovascular {alpha}2-adrenoceptors and angiotensin II receptors in genetic hypertension. Hypertension 38: 353–360, 2001.[Abstract/Free Full Text]
  19. Komlosi P, Fintha A, Bell PD. Current mechanisms of macula densa cell signalling. Acta Physiol Scand 181: 463–469, 2004.[CrossRef][ISI][Medline]
  20. Kornfeld M. Calcium responses in the renal afferent arteriole to angiotensin II and norepinephrine stimulation. In: Department of Physiology and Neuroscience. Lund, Sweden: University of Lund, 1997.
  21. Kornfeld M, Gutierrez AM, Gonzalez E, Salomonsson M, Persson AE. Cell calcium concentration in glomerular afferent and efferent arterioles under the action of noradrenaline and angiotensin II. Acta Physiol Scand 151: 99–105, 1994.[ISI][Medline]
  22. Kurkus J, Thorup C, Persson AE. Renal nerve stimulation restores tubuloglomerular feedback after acute renal denervation. Acta Physiol Scand 164: 237–243, 1998.[CrossRef][ISI][Medline]
  23. Lai EY, Martinka P, Fahling M, Mrowka R, Steege A, Gericke A, Sendeski M, Persson PB, Persson AE, Patzak A. Adenosine restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in renal arterioles. Circ Res 99: 1117–1124, 2006.[Abstract/Free Full Text]
  24. Neylon CB, Summers RJ. [3H]-rauwolscine binding to {alpha} 2-adrenoceptors in the mammalian kidney: apparent receptor heterogeneity between species. Br J Pharmacol 85: 349–359, 1985.[ISI][Medline]
  25. Oberhauser V, Vonend O, Rump LC. Neuropeptide Y and ATP interact to control renovascular resistance in the rat. J Am Soc Nephrol 10: 1179–1185, 1999.[Abstract/Free Full Text]
  26. Patzak A, Lai EY, Mrowka R, Steege A, Persson PB, Persson AE. AT1 receptors mediate angiotensin II-induced release of nitric oxide in afferent arterioles. Kidney Int 66: 1949–1958, 2004.[CrossRef][ISI][Medline]
  27. Perez-Rivera AA, Hlavacova A, Rosario-Colon LA, Fink GD, Galligan JJ. Differential contributions of {alpha}-1 and {alpha}-2 adrenoceptors to vasoconstriction in mesenteric arteries and veins of normal and hypertensive mice. Vascul Pharmacol 46: 373–382, 2007.[CrossRef][ISI][Medline]
  28. Peti-Peterdi J. Calcium wave of tubuloglomerular feedback. Am J Physiol Renal Physiol 291: F473–F480, 2006.[Abstract/Free Full Text]
  29. Poiseuille J. Recherches experimentales sur le mouvement des liquides dans les tubes de tres petits diametres; III. Influence du diametre sur la quantite de liquide qui traverse les tubes de tres petits diametres. Comptes Rendus Acad Sci 11: 1041–1048, 1840.
  30. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231–236, 1994.[CrossRef][Medline]
  31. Tarasova O, Sjoblom-Widfeldt N, Nilsson H. Transmitter characteristics of cutaneous, renal and skeletal muscle small arteries in the rat. Acta Physiol Scand 177: 157–166, 2003.[CrossRef][ISI][Medline]
  32. R-core development Team. R: A language and environment for statistical computing, reference index version 2.1.0. Vienna, Austria: R Foundation for Statistical Computing, 2005.
  33. Walsh MP, Kargacin GJ, Kendrick-Jones J, Lincoln TM. Intracellular mechanisms involved in the regulation of vascular smooth muscle tone. Can J Physiol Pharmacol 73: 565–573, 1995.[ISI][Medline]
  34. Vonend O, Stegbauer J, Sojka J, Habbel S, Quack I, Robaye B, Boeynaems JM, Rump LC. Noradrenaline and extracellular nucleotide cotransmission involves activation of vasoconstrictive P2X(1,3)- and P2Y6-like receptors in mouse perfused kidney. Br J Pharmacol 145: 66–74, 2005.[CrossRef][ISI][Medline]
  35. Yuan BH, Robinette JB, Conger JD. Effect of angiotensin II and norepinephrine on isolated rat afferent and efferent arterioles. Am J Physiol Renal Fluid Electrolyte Physiol 258: F741–F750, 1990.[Abstract/Free Full Text]
  36. Zhao X, Cook AK, Field M, Edwards B, Zhang S, Zhang Z, Pollock JS, Imig JD, Inscho EW. Impaired Ca2+ signaling attenuates P2X receptor-mediated vasoconstriction of afferent arterioles in angiotensin II hypertension. Hypertension 46: 562–568, 2005.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/R2225    most recent
00287.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hultström, M.
Right arrow Articles by Persson, A. E. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hultström, M.
Right arrow Articles by Persson, A. E. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.