Edward W. Inscho

Neuronal nitric oxide synthase modulates rat renal microvascular function

This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 +/- 0.2 microns) and efferent (18.5 +/- 0.2 microns) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-L-thiocitrulline (L-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses to L-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, N omega-nitro-L-arginine (L-NNA) were only attenuated by papillectomy. L-SMTC (10 microM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, L-NNA (100 microM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary micro-vasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 ± 0.2 μm) and efferent (18.5 ± 0.2 μm) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-l-thiocitrulline (l-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses tol-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, N ω-nitro-l-arginine (l-NNA) were only attenuated by papillectomy.l-SMTC (10 μM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, l-NNA (100 μM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary microvasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.

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.

Extracellular ATP in the regulation of renal microvascular function.

Considerable attention has been focused on the purine nucleoside, adenosine, in the control of renal blood flow, epithelial transport, and renin secretion; however, surprisingly little attention has been directed toward the renal effects of purine nucleotides such as adenosine triphosphate (ATP). Recent studies utilizing in vivo micropuncture and in vitro techniques have demonstrated that renal vascular, epithelial, and mesangial cells respond to extracellular ATP via mechanisms distinct from those elicited by adenosine. ATP vasoconstricts afferent but not efferent arterioles whereas adenosine vasoconstricts both vascular segments. Adenosine‐mediated afferent arteriolar vasoconstriction is abolished by adenosine receptor antagonists, whereas the response to ATP is enhanced. ATP‐mediated vasoconstriction reaches a maximum within seconds of exposure while the vasoconstriction induced by adenosine develops more slowly. L‐type calcium channel antagonists such as diltiazem or felodipine prevent the sustained afferent vasoconstriction produced by ATP. Data from micropuncture experiments indicate that peritubular capillary infusion of ATP reduces glomerular pressure and results in marked attenuation of the tubuloglomerular feedback mechanism, which transmits signals from the macula densa to the afferent arteriole. These data support the existence of ATP‐sensitive P2 purinoceptors in the preglomerular microvasculature that contribute to the control of renal vascular function via activation of calcium channels.—Inscho, E. W., Mitchell, K. D., Navar, L. G. Extracellular ATP in the regulation of renal microvascular function. FASEB J. 8: 319‐328; 1994.

Cytochrome P450 and Cyclooxygenase Metabolites Contribute to the Endothelin-1 Afferent Arteriolar Vasoconstrictor and Calcium Responses

Arachidonic acid metabolites contribute to the endothelin-1 (ET-1)-induced decrease in renal blood flow, but the vascular sites of action are unknown. Experiments performed in vitro used the rat juxtamedullary nephron preparation combined with videomicroscopy. The response of afferent arterioles to ET-1 was determined before and after cytochrome P450 (CYP450) or cyclooxygenase (COX) inhibition. Afferent arteriolar diameter averaged 20+/-1 microm (n=17) at a renal perfusion pressure of 100 mm Hg. Superfusion with 0.001 to 10 nmol/L ET-1 caused a graded decrease in diameter of the afferent arteriole. Vessel diameter decreased by 30+/-2% and 41+/-2% in response to 1 and 10 nmol/L ET-1, respectively. The afferent arteriolar response to ET-1 was significantly attenuated during administration of the CYP450 hydroxylase inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), such that afferent arteriolar diameter decreased by 19+/-3% and 22+/-3% in response to 1 and 10 nmol/L ET-1, respectively. COX inhibition also greatly attenuated the vasoconstriction elicited by ET-1, whereas the CYP450 epoxygenase inhibitor N-methylsulfonyl-6-(2-proparglyoxyphenyl) hexanamide enhanced the ET-1-mediated vascular response. Additional studies were performed using freshly isolated smooth muscle cells prepared from preglomerular microvessels. Renal microvascular smooth muscle cells were loaded with the calcium-sensitive dye fura 2 and studied by use of single-cell fluorescence microscopy. Basal renal microvascular smooth muscle cell [Ca(2+)](i) averaged 95+/-3 nmol/L (n=42). ET-1 (10 nmol/L) increased microvascular smooth muscle cell [Ca(2+)](i) to a peak value of 731+/-75 nmol/L before stabilizing at 136+/-8 nmol/L. Administration of DDMS or the COX inhibitor indomethacin significantly attenuated the renal microvascular smooth muscle cell calcium response to ET-1. These data demonstrate that CYP450 hydroxylase and COX arachidonic acid metabolites contribute importantly to the afferent arteriolar diameter and renal microvascular smooth muscle cell calcium responses elicited by ET-1.

Contribution of cytochrome P450 epoxygenase and hydroxylase pathways to afferent arteriolar autoregulatory responsiveness.

Previous studies have demonstrated an important role for the cytochrome P450 (CYT‐P450) pathway in afferent arteriole autoregulatory responses but the involvement of specific pathways remains unknown. Experiments were performed to determine the role of CYT‐P450 epoxygenase and hydroxylase pathways in pressure mediated preglomerular autoregulatory responses. Afferent arteriolar diameter was measured as renal perfusion pressure was increased from 80–160u2003mmHg. Afferent arteriolar diameter averaged 19±2u2003μm at a renal perfusion pressure of 80u2003mmHg and decreased by 15±2% when pressure was increased to 160u2003mmHg. Inhibition of the epoxygenase pathway with 6‐(2‐proparglyloxyphenyl)hexanoic acid (PPOH), enhanced the microvascular response to increasing renal perfusion pressure. In the presence of 50u2003μM PPOH, afferent arteriolar diameter decreased by 29±4% when pressure was increased from 80–160u2003mmHg. Likewise, the sulphonimide derivative of PPOH, N‐methylsulphonyl‐6‐(2‐proparglyloxyphenyl) hexanamide (MS‐PPOH, 50u2003μM), enhanced the afferent arteriolar response to increasing renal perfusion pressure. In contrast, the selective CYT‐P450 hydroxylase inhibitor, N‐methylsulphonyl‐12,12‐dibromododec‐11‐enamide (DDMS) attenuated the vascular response to increasing renal perfusion pressure. In the pressure of 25u2003μM DDMS, afferent arteriolar diameter decreased by 4±2% when pressure was increased from 80–160u2003mmHg. These results suggest that CYT‐P450 metabolites of the epoxygenase pathway alter afferent arteriolar responsiveness and thereby modify the ability of the preglomerular vasculature to autoregulate renal blood flow. Additionally, these results provide further support to the concept that a metabolite of the hydroxylase pathway is an integral component of the afferent arteriolar response to elevations in perfusion pressure.

Afferent and Efferent Arteriolar Vasoconstriction to Angiotensin II and Norepinephrine Involves Release of Ca2+ From Intracellular Stores

Renal vascular responses to angiotensin II (Ang II) and norepinephrine (NE) are reported to involve both mobilization of calcium from intracellular stores and activation of calcium influx pathways. The present study was conducted to determine the contribution of calcium release from intracellular stores to afferent and efferent arteriolar responses to Ang II and NE. Experiments were performed in vitro using the blood-perfused, juxtamedullary nephron technique combined with videomicroscopy. The responses of afferent and efferent arterioles to Ang II and NE were determined before and after depletion of intracellular calcium pools with 1 mumol/L thapsigargin. Positive control responses were obtained with 55 mmol/L KCI. Ang II concentrations of 0.1, 1.0, and 10 nmol/L decreased afferent arteriolar diameter by 10 +/- 4%, 17 +/- 4%, and 29 +/- 6%, respectively (P < .05; n = 8). NE also decreased afferent diameter by 5 +/- 1%, 13 +/- 1%, and 57 +/- 9% at concentrations of 10, 100, and 1000 nmol/L, respectively (P < .05; n = 6). Thapsigargin treatment shifted the afferent arteriolar concentration-response curves for both Ang II and NE significantly to the right. Nevertheless, KCI evoked a pronounced vasoconstriction and decreased afferent diameter by 56 +/- 7% (P < .05; n = 6). Postglomerular responses to Ang II and NE were abolished by thapsigargin. During the control period, efferent diameter decreased by 3 +/- 1%, 7 +/- 2%, and 14 +/- 4% for the three Ang II concentrations and 3 +/- 1%, 5 +/- 1%, and 15 +/- 4% in response to the three NE concentrations, respectively. These responses were completely eliminated in the presence of thapsigargin, whereas KCI evoked an efferent arteriolar vasoconstriction of 57 +/- 9% (P < .05). These data demonstrate that agonist-induced calcium release from intracellular stores represents an essential component in the afferent and efferent arteriolar response to Ang II and NE. Furthermore, they suggest that efferent arteriolar responses to these agents may rely more heavily on calcium release from this store, whereas afferent responses may include activation of other pathways.

Modulation of intracellular calcium and calmodulin by melatonin in MCF-7 human breast cancer cells.

The pineal hormone, melatonin, has been shown to inhibit the proliferation of the estrogen receptor alpha (ERα)‐positive macrophage chemotactic factor (MCF)‐7 human breast cancer cells. Previous studies from other systems indicate that melatonin modulates the calcium (Ca2+)/calmodulin (CaM) signaling pathway either by changing intracellular calcium concentration ([Ca2+]i) via activation of its G‐protein coupled membrane receptors, or through a direct interaction with CaM. In this study, although melatonin alone had no effect on basal [Ca2+]i in MCF‐7 cells, it significantly enhanced the elevation of [Ca2+]i induction by extracellular adenosine triphosphate (ATP), which increases [Ca2+]i via the G protein‐coupled P2y‐purinoceptor and the phospholipase C (PLC) pathway. Pretreatment of MCF‐7 cells with 10–7u2003M melatonin increased the 10–5u2003M ATP‐induced [Ca2+]i peak change from 79.4u2003±u200311.6u2003nM to 146.2u2003±u200322.3u2003nM. Furthermore, without changing total cellular CaM levels, melatonin markedly increased the amount of membrane‐bound CaM to 237 and 162% of control levels after 1 and 6u2003hr of treatment, respectively. Cytosolic CaM levels were also elevated to 172% of control after 6u2003hr of melatonin treatment. Correlative growth studies demonstrated that ATP (10−5u2003M) can stimulate MCF‐7 cell growth, that melatonin can suppress MCF‐7 cell proliferation, but that pretreatment of MCF‐7 cells with melatonin followed by ATP(10–5u2003M), like 10–4u2003M ATP can further suppress MCF‐7 cell proliferation; this indicates that melatonins potentiation of ATP induced [Ca2+]i may be above the threshold for cell growth. Given the important role of [Ca2+]i and CaM in tumor cell homeostasis and proliferation and melatonins modulation of [Ca2+]i, melatonins effects on the Ca2+/CaM signaling pathway may play an important role in mediating the growth‐inhibitory effect of melatonin on MCF‐7 human breast cancer cells.

Juxtamedullary afferent arteriolar responses to P1 and P2 purinergic stimulation.

We assessed the responsiveness of rat juxtamedullary afferent arterioles to purinergic stimulation using the in vitro blood-perfused juxtamedullary nephron technique combined with videomicroscopy to allow direct measurement of arteriolar inside diameter. To minimize the contribution of endogenously formed angiotensin II, all rats were pretreated with enalaprilat (2 mg i.v.) for 3d minutes before the right kidney was isolated and prepared for study. Renal perfusion pressure was set at 110 mm Hg and held constant Afferent arteriolar diameter averaged 20.9±0.8 μm (n=41) under control conditions. Exposure to 1.0 μM 2-chloroadenosine induced a significant (11.1 ±3.2%) reduction in vessel diameter, whereas a 100 &mu:M concentration induced an afferent vasodilation (7.6±1.5%;pμ0.05). These data are consistent with the preferential stimulation of high affinity constrictor adenosine receptors (A1) at lower concentrations and activation of lower affinity vasodilator adenosine receptors (A2) at higher concentrations. In contrast, ATP elicited a significant afferent vasoeonstriction of approximately 9.2%, 12.9%, and 10.0% at concentrations in the range of 1–100 μM (p<0.05). Treatment with ADP, at concentrations up to 100 μM, failed to alter vessel caliber significantly. Furthermore, the nonhydrolyzable ATP analogue α,βmethylene ATP produced a rapid and potent vasoeonstriction, which mimicked the response to ATP. These data reveal the presence of both adenosine-sensitive P1 and ATP-sensitive P2 purinergic receptors on rat juxtamedullary afferent arterioles and demonstrate that ATP can induce afferent arteriolar vasoeonstriction directly without first requiring hydrolysis to adenosine.

Role of Renal Nerves in Afferent Arteriolar Reactivity in Angiotensin-Induced Hypertension

The objective of this study was to determine the contribution of renal nerves to the enhanced afferent arteriolar reactivity observed in angiotensin II (Ang II)-induced hypertension. Uninephrectomized Sprague-Dawley rats were divided into four groups: sham rats, renal-denervated rats, Ang II-infused (at 40 ng/min for 13 days) rats, and Ang II-infused+renal-denervated rats. With the use of an implanted arterial catheter, mean arterial pressure (MAP) was monitored in conscious rats. Ang II infusion resulted in a progressive increase in MAP from 98 +/- 1 (day 0) to 166 +/- 7 mm Hg (day 13). This increase in MAP was attenuated in denervated rats and averaged 136 +/- 3 mm Hg on day 13. Kidneys were harvested on day 13 for microcirculatory experiments or measurement of intrarenal Ang II levels. Basal afferent arteriolar diameter was similar in all groups, and group averages ranged from 19.6 to 20.7 microns. Chronic Ang II infusion increased intrarenal Ang II levels. Renal denervation did not alter this effect. Increasing perfusion pressure from 100 to 160 mm Hg reduced afferent arteriolar diameter significantly by 11.2 +/- 0.6% in the sham group and by a similar degree in the remaining three groups. Superfusion with Ang II (10 nmol/L) reduced afferent arteriolar diameter by 34.3 +/- 2.0% in the sham group. This response was enhanced in Ang II-infused (62.3 +/- 3.4%) but not in renal-denervated or Ang II-infused+renal-denervated rats. Additionally, the enhanced afferent arteriolar reactivity to Ang II was not influenced by adrenergic receptor blockade. The afferent arteriolar response to norepinephrine was enhanced in renal-denervated, Ang II-infused, and Ang II-infused+renal-denervated rats compared with sham controls. Administration of the calcium ionophore A23187 decreased afferent arteriolar diameter similarly in all four groups. These results indicate that renal nerves contribute to the development of hypertension and to the enhanced afferent arteriolar responsiveness to Ang II elicited by chronic Ang II infusion.

Purinoceptor-Mediated Calcium Signaling in Preglomerular Smooth Muscle Cells

-The current studies were performed to determine the contribution of calcium mobilization and voltage-dependent calcium influx to the increase in [Ca2+]i elicited by ATP and UTP. Suspensions of freshly isolated smooth muscle cells were prepared from preglomerular microvessels by enzymatic digestion and loaded with the Ca2+-sensitive dye fura 2. The effect of ATP and UTP on [Ca2+]i was studied on single cells with standard microscope-based fluorescence photometry techniques. Resting [Ca2+]i averaged 80+/-3 nmol/L (n=219 single cells from 58 dispersions). ATP (100 micromol/L) increased [Ca2+]i to a peak value of 845+/-55 nmol/L (n=70 single cells from 38 dispersions) before stabilizing at 124+/-81 nmol/L. Similarly, 100 micromol/L UTP (n=39 single cells from 26 dispersions) stimulated a peak increase in [Ca2+]i of 1426+/-584 nmol/L before reaching a stable plateau of 123+/-10 nmol/L. The [Ca2+]i response to ATP and UTP was also assessed in the absence of extracellular calcium. In these studies, exposure to 100 micromol/L ATP induced a transient peak increase in [Ca2+]i, with the plateau phase being totally abolished. In contrast, exposure to 100 micromol/L UTP under calcium-free conditions resulted in no detectable change in the UTP-mediated increase in [Ca2+]i. The role of L-type calcium channels in the response was assessed with the calcium channel antagonist diltiazem. Incubation with diltiazem (10 micromol/L) markedly reduced the response to ATP, whereas the response to UTP was only slightly reduced. These data demonstrate that both ATP and UTP directly stimulate a biphasic increase in [Ca2+]i in renal microvascular smooth muscle cells. Furthermore, the data suggest that the elevation of [Ca2+]i elicited by ATP is largely dependent on calcium influx through L-type calcium channels, whereas the response to UTP appears to derive primarily from mobilization of calcium from intracellular stores.