Afferent arteriolar vasodilation to the sulfonimide analog of 11, 12-epoxyeicosatrienoic acid involves protein kinase A.

The current study determined the contribution of protein kinase-A (PKA) and protein kinase-G (PKG) to the vasodilation elicited by the N-methylsulfonimide analog of 11,12-epoxyeicosatrienoic acid (11, 12-EET). Experiments were performed, in vitro, using the juxtamedullary nephron preparation combined with videomicroscopy. The response of afferent arterioles to the sulfonimide analog of 11, 12-EET, was determined before and after inhibition of PKA, PKG, or guanylyl cyclase. Afferent arterioles, preconstricted with 0.5 micromol/L norepinephrine, averaged 18+/-1 microm (n=25) at a renal perfusion pressure of 100 mm Hg. Superfusion with 0.01 to 100 nmol/L of the 11,12-EET analog caused a graded increase in diameter of the afferent arteriole. Vessel diameter increased by 11+/-1% and 15+/-1%, respectively, in response to 10 and 100 nmol/L of the 11,12-EET analog. The afferent arteriolar response to 10 and 100 nmol/L of the 11,12-EET analog was significantly attenuated during inhibition of PKA with 10 micromol/L H-89 (n=7) or 5 micromol/L myristolated PKI (n=6), such that afferent arteriolar diameter increased by only 5+/-2% and 2+/-1%, respectively, in response to 100 nmol/L of the 11, 12-EET analog. In contrast, the afferent arteriolar vasodilatory response to the 11,12-EET analog was unaffected by PKG or guanylyl cyclase inhibition. In the presence of 200 micromol/L histone H2B (n=5) or 10 micromol/L ODQ (n=7), the afferent arteriolar diameter increased by 16+/-3% and 12+/-2%, respectively, in response to 100 nmol/L of the 11,12-EET analog. These results demonstrate that activation of PKA is an important mechanism responsible for the afferent arteriolar vasodilation elicited by the sulfonimide analog of 11,12-EET.

Afferent arteriolar responses to ANG II involve activation of PLA2 and modulation by lipoxygenase and P-450 pathways.

Activation of angiotensin receptors activates phospholipase A2 (PLA2) in various tissues, resulting in the release of arachidonic acid and formation of vasoactive metabolites. The present study examined the role of the lipoxygenase and cytochrome P-450 pathways by evaluating the effects of PLA2, cyclooxygenase, lipoxygenase, and epoxygenase inhibition on the afferent arteriolar responses to angiotensin II (ANG II) and norepinephrine in the vitro perfused rat juxtamedullary nephron preparation. ANG II (0.01-100 nM) resulted in a dose-dependent afferent arteriolar vasoconstriction ranging from 3 +/- 1 to 32 +/- 2% (n = 47). Norepinephrine at 0.01, 0.1, and 1.0 microM also decreased afferent arteriolar diameter by 5 +/- 1, 17 +/- 1, and 34 +/- 2%, respectively (n = 43). In the presence of arachidonyl trifluoromethyl ketone (AACOCF3, 20 microM), a PLA2 inhibitor, afferent arteriolar vasoconstriction to ANG II (100 nM) was attenuated, and the diameter decreased by 23 +/- 4% (n = 7). The cyclooxygenase inhibitor, indomethacin (10 microM), and the cyclooxygenase-2 inhibitor, NS-398 (10 microM), did not affect the afferent arteriolar response to ANG II. The lipoxygenase inhibitor biacalein (1 microM) attenuated the afferent arteriolar response to ANG II, and vessel diameter decreased by 11 +/- 5% (n = 6) in response to 100 nM ANG II. On the other hand, miconazole (1 microM), a selective epoxygenase inhibitor, enhanced the afferent arteriolar vasoconstriction to 100 nM ANG II. 17-Octadecynoic acid (17-ODYA, 1 microM), an inhibitor of hydroxylase and epoxygenase metabolism of arachidonic acid, also increased the responsiveness of the afferent arteriole. PLA2, lipoxygenase, or cytochrome P-450 inhibition had no effect on the afferent arteriolar vasoconstriction to norepinephrine. The afferent arteriolar vasoconstrictor response to norepinephrine (0.1 microM) was enhanced by indomethacin or NS-398, and diameter decreased by 25 +/- 3% and 28 +/- 4%, respectively. Results of this study suggest that metabolites of the cyclooxygenase pathway attenuate the afferent arteriolar vasoconstrictor effect of norepinephrine. Furthermore, these data suggest that activation of PLA2 is involved in part of the afferent arteriolar response to ANG II and that metabolites of the lipoxygenase pathway augment and metabolites of the epoxygenase pathway attenuate the afferent arteriolar vasoconstrictor effect of ANG II.

Candesartan cilexetil protects against loss of autoregulatory efficiency in angiotensin II-infused rats.

Renal autoregulatory efficiency is compromised in angiotensin-II (AngII)-dependent Goldblatt hypertension. The current studies were performed to assess renal autoregulatory capability in AngII-infused hypertensive rats and to determine the effect of chronic candesartan cilexetil treatment on autoregulatory behavior. Rats received chronic infusion of AngII (60 ng/min) or vehicle via an osmotic minipump implanted subcutaneously in the dorsum of the neck. Selected rats received the novel AT1 receptor blocker candesartan cilexetil (1.0 mg/kg per d) in the drinking water. Systolic BP averaged 118+/-1 mmHg (n=34) before pump implantation. Chronic AngII infusion for 6 d increased arterial pressure to 151+/-4 mmHg. Candesartan cilexetil administration prevented the AngII-dependent increase in systolic BP. Microvascular autoregulation experiments were performed in vitro using the blood-perfused juxtamedullary nephron technique combined with videomicroscopy. Renal perfusion pressure was set at 100 mmHg during the control period before being decreased to 65 mmHg. Afferent arteriolar diameter was measured continuously as the perfusion pressure was increased from 65 mmHg to 170 mmHg in 15-mmHg increments. Afferent arteriolar diameter in sham-treated rats was 120% of control at a perfusion pressure of 65 mmHg and decreased to 76% of the control diameter at 170 mmHg (n=6). This behavior is consistent with normal autoregulatory behavior. Arterioles from rats receiving chronic infusion of AngII exhibited compromised renal microvascular autoregulatory efficiency. Afferent arteriolar diameter in AngII-treated kidneys varied from 103 to 100% (n=6) of the control diameter over the same pressure range of 65 to 170 mmHg. This blunting of autoregulatory behavior was prevented by AT1 receptor blockade. In animals receiving AngII + candesartan cilexetil, stepwise changes in perfusion pressure elicited changes in afferent arteriolar diameter between 120 and 84% after 6 d of treatment (n=6). These data suggest that chronic elevations in circulating AngII and/or the associated increase in arterial pressure impairs renal autoregulatory capability. Furthermore, inhibition of AT1 receptors with candesartan cilexetil provides protection against AngII-mediated increases in arterial pressure and prevents the associated deterioration of renal autoregulatory responsiveness.

ATP-mediated Ca2+ signaling in preglomerular smooth muscle cells.

We performed studies to determine the effect of extracellular ATP on the intracellular Ca2+ concentration ([Ca2+]i) in freshly isolated microvascular smooth muscle cells (MVSMC). Suspensions of preglomerular MVSMC were prepared by enzymatic digestion and loaded with fura 2. Single cells were studied using a microscope-based fluorescence spectrophotometer during superfusion of a physiological salt solution with 1.8 mM Ca2+ and during exposure to similar solutions containing ATP. Under control conditions, baseline [Ca2+]i averaged 107 +/- 6 nM (n = 86 cells from 34 animals). ATP administration elicited concentration-dependent increases in [Ca2+]i. Exposure to ATP concentrations of 1, 10, and 100 microM increased intracellular Ca2+ to peak concentrations of 133 +/- 20, 338 +/- 37, and 367 +/- 35 nM, respectively (P < 0.05 vs. respective baseline). Steady-state [Ca2+]i increased to 113 +/- 15, 150 +/- 16 (P < 0.05 vs. baseline), and 180 +/- 12 nM (P < 0.05 vs. baseline) for the same groups. The [Ca2+]i response to ATP was also assessed in the absence of extracellular Ca2+ and during blockade of L-type Ca2+ channels with diltiazem. In these studies, exposure to 100 microM ATP induced a transient peak increase in [Ca2+]i with the plateau phase being totally abolished under Ca2+-free conditions and markedly attenuated during Ca2+ channel blockade, respectively. These data indicate that ATP-mediated P2-receptor activation increases [Ca2+]i in freshly isolated preglomerular MVSMC by stimulating Ca2+ release from intracellular stores, in addition to stimulating the influx of extracellular Ca2+ through voltage-gated L-type Ca2+ channels.

Agonist-induced calcium regulation in freshly isolated renal microvascular smooth muscle cells.

The studies presented here were performed to determine the effect of agonist stimulation on the cytosolic free Ca2+ concentration ([Ca2+]i) in single smooth muscle cells, freshly isolated from afferent arterioles and interlobular arteries averaging between 10 to 40 microns in diameter. Microvessels were obtained from male Sprague-Dawley rats using an iron oxide collection technique followed by collagenase digestion. Freshly isolated microvascular smooth muscle cells (MVSMC) were loaded with fura 2 and studied using fluorescence photometry techniques. The resting [Ca2+]i averaged 67 +/- 3 nM (N = 82 cells). Increasing the extracellular K+ concentration significantly increased [Ca2+]i dose-dependently (P < 0.05). Involvement of extracellular Ca2+ in the response to KCl-induced depolarization was also evaluated. Resting [Ca2+]i increased approximately 132% from 40 +/- 5 nM to 93 +/- 26 nM in response to 90 mM extracellular KCl. This change was abolished in nominally Ca(2+)-free conditions and markedly attenuated by diltiazem. Inhibition of K+ channels with charybdotoxin or tetraethylammonium chloride produced a modest transient increase in [Ca2+]i during the response to 30 mM K+ and had no detectable effect on responses to 90 mM K+. Studies were also performed to establish whether freshly isolated renal MVSMC exhibit appropriate responses to receptor-dependent physiological agonists. Angiotensin II (100 nM) increased cell Ca2+ from 97 +/- 10 nM to 265 +/- 47 nM (N = 12 cells). Similarly, 100 microM ATP increased MVSMC [Ca2+]i from a control level of 71 +/- 14 nM to 251 +/- 47 nM (N = 11 cells). Norepinephrine administration caused [Ca2+]i to increase from 63 +/- 4 nM to 212 +/- 47 nM (N = six cells), and vasopressin increased [Ca2+]i from 86 +/- 10 nM to 352 +/- 79 nM (N = five cells). These data demonstrate that receptor-dependent and -independent vasoconstrictor agonists increase [Ca2+]i in MVSMC, freshly isolated from rat preglomerular vessels. Furthermore, the ability to measure [Ca2+]i in responses to physiological stimuli in these single cells permits investigation of signal transduction mechanisms involved in regulating renal microvascular resistance.