Biomedical subjects

J D Imig

Publications and source records attributed to J D Imig.

At least 19 recordsLinked to original sources

PubMed2006

Increased renal vascular reactivity to ANG II after unilateral nephrectomy in the rat involves 20-HETE.

This study examined the role of intrarenal ANG II in the renal vascular reactivity changes occurring in the remaining kidney undergoing adaptation following contralateral nephrectomy. Renal blood flow responses to intrarenal injections of ANG II (0.25 to 5 ng) were measured in anesthetized euvolemic male Wistar rats 1, 4, 12, and 24 wk after uninephrectomy (UNX) or sham procedure (SHAM). At week 4, renal vasoconstriction induced by 2 ng ANG II was greater in UNX (69 +/- 5%) than in SHAM rats (50 +/- 3%; P < 0.01). This response was inhibited, by 50 and 66%, and by 20 and 25%, in SHAM and UNX rats, after combined injections of ANG II and losartan, or PD-123319 (P < 0.05), respectively. Characteristics of ANG II receptor binding in isolated preglomerular resistance vessels were similar in the two groups. After prostanoid inhibition with indomethacin, renal vasoconstriction was enhanced by 42 +/- 8% (P < 0.05), only in SHAM rats, whereas after 20-HETE inhibition with HET0016, it was reduced by 53 +/- 16% (P < 0.05), only in UNX rats. These differences vanished after concomitant prostanoid and 20-HETE inhibition in the two groups. After UNX, renal cortical protein expression of cytochrome P-450 2c23 isoform (CYP2c23) and cyclooxygenase-1 (COX-1) was unaltered, but it was decreased for CYP4a and increased for COX-2. In conclusion, renal vascular reactivity to ANG II was significantly increased in the postuninephrectomy adapted kidney, independently of protein expression, but presumably involving interactions between 20-HETE and COX in the renal microvasculature and changes in the paracrine activity of ANG II and 20-HETE.

Adaptation, Physiological↗

PubMed2004

Renal autoregulation in P2X1 knockout mice.

Autoregulation of renal blood flow is an established physiological phenomenon, however the signalling mechanisms involved remain elusive. Autoregulatory adjustments in preglomerular resistance involve myogenic and tubuloglomerular feedback (TGF) influences. While there is general agreement on the participation of these two regulatory pathways, the signalling molecules and effector mechanisms have not been identified. Currently, there are two major hypotheses being considered to explain the mechanism by which TGF signals are transmitted from the macula densa to the afferent arteriole. The adenosine hypothesis proposes that the released adenosine triphosphate (ATP) is hydrolysed to adenosine and this product stimulates preglomerular vasoconstriction by activation of A(1) receptors on the afferent arteriole. Alternatively, the P2 receptor hypothesis postulates that ATP released from the macula densa directly stimulates afferent arteriolar vasoconstriction by activation of ATP-sensitive P2X(1) receptors. This hypothesis has emerged from the realization that P2X(1) receptors are heavily expressed along the preglomerular vasculature. Inactivation of P2X(1) receptors impairs autoregulatory responses while afferent arteriolar responses to A(1) adenosine receptor activation are retained. Autoregulatory behaviour is markedly attenuated in mice lacking P2X(1) receptors but responses to adenosine A(1) receptor activation remain intact. More recent experiments suggest that P2X(1) receptors play an essential role in TGF-dependent vasoconstriction of the afferent arteriole. Interruption of TGF-dependent influences on afferent arteriolar diameter, by papillectomy or furosemide treatment, significantly attenuated pressure-mediated afferent arteriolar vasoconstriction in wild-type mice but had no effect on the response in P2X(1) knockout mice. Collectively, these observations support an essential role for P2X(1) receptors in TGF-mediated afferent arteriolar vasoconstriction.

Adenosine Triphosphate↗

PubMed2003

Kidney CYP450 enzymes: biological actions beyond drug metabolism.

Arachidonic acid can be metabolized by cytochrome p450 (CYP450) enzymes to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosa-trienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These arachidonic acid metabolites are involved in the regulation of renal epithelial transport and vascular function. 20-HETE and EETs are produced in the renal microvascular smooth muscle cells and endothelial cells, respectively. 20-HETE constricts the preglomerular arterioles by inhibiting K(+) channels, and contributes importantly to renal blood flow autoregulatory responsiveness of the afferent arterioles. EETs dilate the preglomerular arterioles by activating the renal smooth muscle cell Ca(2+)-activated K(+) channels and hyperpolarizing smooth muscle cells. These EET actions are consistent with their identification as endothelium-derived hyperpolarizing factors (EDHFs). In the kidney, EETs and 20-HETE are also produced in the proximal tubule and the thick ascending loop of Henle, and these metabolites modulate ion transport in the proximal tubules and the thick ascending limb by inhibiting Na(+)-K(+)-ATPase and the Na(+)-K(+)-2Cl(-) cotransporter. CYP450 metabolites also act as second messengers for many paracrine and hormonal agents, including endothelin, nitric oxide, and angiotensin II. The production of kidney CYP450 arachidonic acid metabolites is altered in diabetes, pregnancy, hepatorenal syndrome, and in various models of hypertension, and it is likely that changes in this system contribute to the abnormalities in renal function that are associated with many of these conditions.

Animals↗

PubMed2001

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension.

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (-/-) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (-/-) mice and minimizes Cyp 4a12 expression and arachidonate omega-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (-/-) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 omega-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Androgens↗

PubMed2001

Enhanced renal microvascular reactivity to angiotensin II in hypertension is ameliorated by the sulfonimide analog of 11,12-epoxyeicosatrienoic acid.

OBJECTIVES: Epoxygenase metabolites produced by the kidney affect renal blood flow and tubular transport function and 11,12-epoxyeicosatrienoic acid (11,12-EET) has been putatively identified as an endothelium-derived hyperpolarizing factor. The current studies were performed to determine the influence of 11,12-EET on the regulation of afferent arteriolar diameter in angiotensin II-infused hypertensive rats. MATERIALS AND METHODS: Male Sprague-Dawley rats received angiotensin II (60 ng/min) or vehicle via an osmotic minipump. Angiotensin II-infused hypertensive and vehicle-infused normotensive rats were studied for 2 weeks following implantation of the minipump. Renal microvascular responses to the sulfonimide analog of 11,12-EET (11,12-EET-SI) and angiotensin II were observed utilizing the in-vitro juxtamedullary nephron preparation. Renal cortical epoxygenase enzyme protein levels were quantified by Western blot analysis. Renal microvessels were also isolated and epoxygenase metabolite levels measured by negative ion chemical ionization (NICI)/gas chromatography-mass spectroscopy. RESULTS: Systolic blood pressure averaged 118 +/- 2 mmHg prior to pump implantation and increased to 185 +/- 7 mmHg in rats infused with angiotensin II for 2 weeks. Afferent arteriolar diameters of 2-week normotensive animals averaged 22 +/- 1 microm. Diameters of the afferent arterioles were 17% smaller in hypertensive rats (P< 0.05); however, arterioles from both groups responded to 11,12-EET-SI (100 nmol) with similar 15-17% increases in diameter. As we previously demonstrated, the afferent arteriolar reactivity to angiotensin II was enhanced in angiotensin II-infused animals. Interestingly, elevation of 11,12-EET-SI levels to 100 nmol reversed the enhanced vascular reactivity to angiotensin II associated with angiotensin II hypertension. Renal microvascular EET levels were not different between groups and averaged 81 +/- 9 and 87 +/- 13 pg/mg per 30 min in normotensive and hypertensive animals, respectively. Renal cortical microsomal levels of the epoxygenase CYP2C23 and CYP2C11 proteins were also similar in normotensive and angiotensin II hypertensive rats. CONCLUSIONS: Taken together, these data support the concept that renal microvascular 11,12-EET activity and levels may not properly offset the enhanced angiotensin II renal vasoconstriction during angiotensin II hypertension.

8,11,14-Eicosatrienoic Acid↗

PubMed2001

The CYP450 hydroxylase pathway contributes to P2X receptor-mediated afferent arteriolar vasoconstriction.

This study was conducted to test the hypothesis that the cytochrome P-450 (CYP450) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the afferent arteriolar response to P2 receptor activation. Afferent arteriolar responses to ATP, the P2X agonist, alpha,beta-methylene ATP and the P2Y agonist UTP were determined before and after treatment with the selective CYP450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE). Stimulation with 1.0 and 10 microM ATP elicited an initial preglomerular vasoconstriction of 12 +/- 1% and 45 +/- 4% and a sustained vasoconstriction of 11 +/- 1% and 11 +/- 2%, respectively. DDMS or 20-HEDE significantly attenuated the sustained afferent arteriolar constrictor response to ATP. alpha,beta-Methylene ATP (1 microM) induced a rapid initial afferent vasoconstriction of 64 +/- 3%, which partially recovered to a stable diameter 10 +/- 1% smaller than control. Both DDMS and 20-HEDE significantly attenuated the initial vasoconstriction and abolished the sustained vasoconstrictor response to alpha,beta-methylene ATP. UTP decreased afferent diameter by 50 +/- 5% and 20-HEDE did not change this response. In addition, the ATP-induced increase in the intracellular Ca2+ concentration in preglomerular microvascular smooth muscle cells was significantly attenuated by 20-HEDE. Taken together, these results are consistent with the hypothesis that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors.

Adenosine Triphosphate↗

PubMed2001

Calcium signaling pathways utilized by P2X receptors in freshly isolated preglomerular MVSMC.

This study tested the hypothesis that P2X receptor activation increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in preglomerular microvascular smooth muscle cells (MVSMC) by evoking voltage-dependent calcium influx. MVSMC were obtained and loaded with the calcium-sensitive dye fura 2 and studied by using single-cell fluorescence microscopy. The effect of P2X receptor activation on [Ca(2+)](i) was assessed by using the P2X receptor-selective agonist alpha,beta-methylene-ATP and was compared with responses elicited by the endogenous P2 receptor agonist ATP. alpha,beta-Methylene-ATP increased [Ca(2+)](i) dose dependently. Peak increases in [Ca(2+)](i) averaged 37 +/- 11, 73 +/- 15, and 103 +/- 21 nM at agonist concentrations of 0.1, 1, and 10 microM, respectively. The average peak response elicited by 10 microM alpha,beta-methylene-ATP was approximately 34% of the response obtained with 10 microM ATP. alpha,beta-Methylene-ATP induced a transient increase in [Ca(2+)](i) before [Ca(2+)](i) returned to baseline, whereas ATP induced a biphasic response including a peak response followed by a sustained plateau. In Ca(2+)-free medium, ATP induced a sharp transient increase in [Ca(2+)](i), whereas the response to alpha,beta-methylene-ATP was abolished. Ca(2+) channel blockade with 10 microM diltiazem or nifedipine attenuated the response to alpha,beta-methylene-ATP, whereas nonspecific blockade of Ca(2+) influx pathways with 5 mM Ni(2+) abolished the response. Blockade of P2X receptors with the novel P2X receptor antagonist NF-279 completely but reversibly abolished the response to alpha,beta-methylene-ATP. These results indicate that P2X receptor activation by alpha,beta-methylene-ATP increases [Ca(2+)](i) in preglomerular MVSMC, in part, by stimulating voltage-dependent Ca(2+) influx through L-type Ca(2+) channels.

Adenosine Triphosphate↗

PubMed2000

Postglomerular vasoconstriction to angiotensin II and norepinephrine depends on intracellular calcium release.

The current study was performed to determine the effect of calcium store depletion with cyclopiazonic acid (CPA) on the pre- and postglomerular vasoconstrictor responses to angiotensin II (ANG II) and norepinephrine (NE). CPA treatment significantly attenuated the afferent arteriolar response to 10 nM ANG II by 51% and to 1000 nM NE by 19%. Efferent arteriolar responses to ANG II and NE were also greatly attenuated in the presence of CPA. These data demonstrate that afferent and efferent arteriolar responses to ANG II and NE depend on release of calcium from CPA-sensitive intracellular stores. Furthermore, the postglomerular response to these agents exhibits a greater dependency on calcium release from intracellular stores.

Angiotensin II↗

PubMed2000

Afferent arteriolar reactivity to angiotensin II is enhanced during the early phase of angiotensin II hypertension.

Increased renal microvascular reactivity may contribute to the blunted pressure natriuretic response and increase in blood pressure during the development of angiotensin II hypertension. The current studies were performed to determine renal microvascular reactivity during the early phases of angiotensin II-infused hypertension. Male-Sprague Dawley rats received angiotensin II (60 ng/min) or vehicle via an osmotic minipump. Normotensive and angiotensin II hypertensive rats were studied 1 and 2 weeks after implantation of the minipump. Systolic blood pressure averaged 117 +/- 4 mm Hg (n = 31) before pump implantation. Angiotensin II infusion increased systolic blood pressure to 149 +/- 3 and 187 +/- 5 mm Hg on infusion days 6 and 12, respectively. Renal microvascular responses to angiotensin II and norepinephrine at renal perfusion pressures of 100 and 150 mm Hg were observed using the in vitro juxtamedullary nephron preparation. Afferent arteriolar diameters of 1-week normotensive animals averaged 22 +/- 1 microm and after 2 weeks of vehicle infusion averaged 21 +/- 1 microm at a perfusion pressure of 100 mm Hg. In animals infused with angiotensin II for 1 or 2 weeks, diameters of the afferent arterioles perfused at a pressure of 100 mm Hg were 20% and 9% smaller, respectively. Additionally, 1- and 2-week hypertensive animals had an enhanced responsiveness of the renal microvasculature to angiotensin II. At a perfusion pressure of 100 mm Hg, angiotensin II (10 nmol/L) decreased afferent arteriolar diameter by 26 +/- 5% and 22 +/- 3% in the 1- and 2-week angiotensin II hypertensive rats, respectively. In 1- and 2-week normotensive animals, angiotensin II (10 nmol/L) decreased afferent arteriolar diameter by 18 +/- 2% and 15 +/- 2%, respectively, at a perfusion pressure of 100 mm Hg. In contrast, the afferent arteriolar response to norepinephrine was not altered in angiotensin II hypertensive rats. These data demonstrate an elevated renal microvascular resistance and enhanced vascular reactivity that is selective for angiotensin II in the early phases of hypertension development after infusion of angiotensin II. Thus, an alteration in renal microvascular function contributes to the blunted pressure natriuretic response and progressive development of hypertension.

Angiotensin II↗

PubMed2000

Renal responses to AT1 receptor blockade.

Because of the importance of the renin-angiotensin system in the pathophysiology of hypertension and in mediating associated alterations in renal function, angiotensin II (Ang II) AT1 receptor blockers provide a direct means of protecting against influences of excessive Ang II levels. The kidney is an important site of action of Ang II AT1 receptor blockers because intrarenal Ang II not only vasoconstricts the renal vasculature but also reduces sodium excretion and suppresses the pressure natriuresis relationship. Even in normal conditions, intrarenal Ang II content is greater than can be explained on the basis of circulating Ang II and is compartmentalized with proximal tubule concentrations of Ang I and Ang II being several times higher than plasma concentrations. The localization of angiotensinogen in proximal tubule cells further supports the concept that the proximal tubule secretes Ang II or precursors of Ang II into the tubular fluid to activate luminal Ang II receptors. Recent immunohistochemical studies have demonstrated an abundance of AT1 receptors on the luminal surface of proximal and distal tubule cells as well as on vascular smooth muscle cells of afferent and efferent arterioles and on glomerular mesangial cells. Activation of luminal AT1 receptors stimulates the sodium hydrogen exchanger and increases reabsorption rate. The prominence of AT1 receptors in vascular and epithelial tissues in the kidney provides the basis for the powerful effects of AT1 receptor blockers on renal function especially in hypertensive conditions. In the two-kidney, one-clip (2K1C) Goldblatt hypertensive rat model, the nonclipped kidney is renin depleted but the intrarenal Ang II levels are not suppressed and Ang II concentrations in proximal tubular fluid remain high (10(-8) mol/L). AT1 receptor blockers such as candesartan have been shown to cause significant increases in glomerular filtration rate, renal blood flow and proportionately much greater increases in sodium excretion and fractional sodium excretion. Ang II blockade also markedly increases the slope of the pressure natriuresis relationship. The collective actions of Ang II blockers on tubular transport and renal hemodynamics provide long-term effects to regulate sodium balance, which contributes to the long-term control of hypertension.

Angiotensin Receptor Antagonists↗

PubMed2000

Nitric oxide-angiotensin II interactions in angiotensin II-dependent hypertension.

Many studies indicate that renal haemodynamic function in angiotensin II- (ANG II) dependent hypertension is not reduced as much as would be predicted from the elevated ANG II levels suggesting that counteracting renoprotective mechanisms are activated. One important renoprotective effect is mediated by increased levels of nitric oxide. Recent studies using the ANG II-infused hypertensive rat model have shown that inhibition of nitric oxide synthesis (NOS) causes greater decreases in renal blood flow and glomerular filtration rate in ANG II-infused hypertensive rats than in control rats. This augmented nitric oxide-dependent influence is localized primarily in the cortex and to the preglomerular vasculature. The differential effects on the renal cortex and medulla are also reflected by the differences in NOS activities and protein expression. Ca2+-dependent NOS activity was significantly greater in the cortex but not the medulla of the ANG II-infused hypertensive rats compared with control rats. This was associated with marked activation of endothelial NOS protein levels and smaller increases in neuronal NOS protein levels in the cortex but not in the medulla. In contrast, the Ca2+-independent NOS activity and the inducible NOS protein levels in the cortex were significantly lower in the ANG II-infused hypertensive rats. These data support the hypothesis that cortical Ca2+-dependent NOS, primarily endothelial NOS, is stimulated during the early phases of ANG II-induced hypertension and exerts a renoprotective effect on cortical haemodynamics.

Angiotensin II↗

PubMed2000

Early diabetes mellitus stimulates proximal tubule renin mRNA expression in the rat.

BACKGROUND: Enhanced intrarenal angiotensin II (Ang II) activity may contribute to diabetic nephropathy. The proximal tubule is a proposed site of significant intrarenal Ang II production. We determined the effect of early diabetes on mRNA expression of components of the proximal tubule renin-angiotensin system. METHODS: Three groups of male Sprague-Dawley rats were studied after two weeks: (1) control (C), (2) streptozotocin-induced diabetes (STZ), and (3) STZ-induced diabetes, with normoglycemia maintained by insulin implants (STZ-I). Competitive reverse transcription-polymerase chain reaction was used to assay mRNA for renin, angiotensinogen, and angiotensin-converting enzyme in suspensions of proximal tubules; plasma and kidney levels of Ang II were measured by radioimmunoassay, and Western analysis of Ang II subtype 1 (AT1) receptors was performed. RESULTS: STZ rats tended to have increased plasma and intrarenal levels of Ang II compared with C and STZ-I rats. In proximal tubules, mRNA for renin was significantly increased in STZ rats, with reversal to control values in STZ-I rats (C, 2432 +/- 437 vs. STZ, 5688 +/- 890 fg/0.25 microg RNA, P < 0.05 vs. C, N = 9, vs. STZ-I, 1676 +/- 376 fg/0.25 microg RNA, P = NS vs. C). In STZ rats, the AT1 receptor antagonist losartan caused a further fivefold increase in proximal tubule renin mRNA, associated with proximal tubular renin immunostaining. STZ had no significant effect on mRNA expression for angiotensinogen or angiotensin-converting enzyme in proximal tubules. By Western blot analysis, cortical and proximal tubule AT1 receptor protein expression was significantly decreased in STZ rats. CONCLUSIONS: These data suggest activation of the proximal tubule renin-angiotensin system in early STZ diabetes, mediated at least partly by enhanced expression of renin mRNA. Increased local production of Ang II could contribute to tubulointerstitial injury in this model.

Angiotensin II↗

PubMed2000

Eicosanoid regulation of the renal vasculature.

Even though it has been recognized that arachidonic acid metabolites, eicosanoids, play an important role in the control of renal blood flow and glomerular filtration, several key observations have been made in the past decade. One major finding was that two distinct cyclooxygenase (COX-1 and COX-2) enzymes exist in the kidney. A renewed interest in the contribution of cyclooxygenase metabolites in tubuloglomerular feedback responses has been sparked by the observation that COX-2 is constitutively expressed in the macula densa area. Arachidonic acid metabolites of the lipoxygenase pathway appear to be significant factors in renal hemodynamic changes that occur during disease states. In particular, 12(S)- hydroxyeicosatetraenoic acid may be important for the full expression of the renal hemodynamic actions in response to angiotensin II. Cytochrome P-450 metabolites have been demonstrated to possess vasoactive properties, act as paracrine modulators, and be a critical component in renal blood flow autoregulatory responses. Last, peroxidation of arachidonic acid metabolites to isoprostanes appears to be involved in renal oxidative stress responses. The recent developments of specific enzymatic inhibitors, stable analogs, and gene-disrupted mice and in antisense technology are enabling investigators to understand the complex interplay by which eicosanoids control renal blood flow.

Animals↗

PubMed2000

Endothelin-mediated calcium signaling in preglomerular smooth muscle cells.

This study was performed to test the hypothesis that endothelin peptides differentially influence intracellular calcium concentration ([Ca(2+)](i)) in preglomerular microvascular smooth muscle cells (MVSMC), in part through activation of endothelin (ET)(A) receptors. Experiments were performed in vitro with the use of single MVSMC freshly isolated from rat preglomerular microvessels. The effect of ET-1, ET-2, and ET-3 on [Ca(2+)](i) was measured with the use of the calcium-sensitive dye, fura 2, and standard fluorescence microscopy techniques. Baseline [Ca(2+)](i) averaged 84+/-3 nmol/L (n=141 cells from 23 dispersions). ET-1 concentrations of 1, 10, and 100 nmol/L evoked peak increases in [Ca(2+)](i) of 48+/-16, 930+/-125, and 810+/-130 nmol/L, respectively. The time course of the [Ca(2+)](i) response was biphasic, beginning with a rapid initial increase followed by a sustained plateau phase or a period during which [Ca(2+)](i) oscillated sharply. Similar responses were observed after ET-2 administration. In contrast, ET-3 stimulated monophasic increases in [Ca(2+)](i) of only 14+/-5, 33+/-16, and 44+/-19 nmol/L at peptide concentrations of 1, 10, and 100 nmol/L, respectively. These responses are significantly smaller than responses to ET-1 or ET-2, respectively. The relative contributions of calcium mobilization and calcium influx in the response to ET-1 were also evaluated. Removal of calcium from the bathing medium did not significantly alter the peak response to 10 nmol/L ET-1 but abolished the late phase elevation of [Ca(2+)](i). These data demonstrate that endothelin peptides increase [Ca(2+)](i) in preglomerular MVSMC. The concentration-response profiles are consistent with the response involving activation of ET(A) receptors. Furthermore, these results suggest that ET-1 increases [Ca(2+)](i) by stimulating both the release of intracellular calcium and the influx of calcium from the extracellular medium.

Animals↗

PubMed2000

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.

Amides↗

PubMed2000

Epoxygenase metabolites. Epithelial and vascular actions.

Cytochrome P450 (CYP450) epoxygenase enzymes demonstrate organ and cell specific expression and each CYP450 enzyme isoform produces a distinct pattern of epoxyeicosatrienoic acids (EETs). Investigations are beginning to describe the regulation of specific tissue CYP450 epoxygenase isoforms that may be associated with alterations in organ function that occur during various physiological and pathophysiological states. The main biological actions of EETs are their ability to affect epithelial ion transport and vascular smooth muscle cell function. This chapter focuses on the organ localization and production of EETs and the action of EETs on epithelial and vascular smooth muscle cells.

Arachidonic Acids↗

PubMed1999

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 80-160 mmHg. Afferent arteriolar diameter averaged 19+/-2 microm at a renal perfusion pressure of 80 mmHg and decreased by 15+/-2% when pressure was increased to 160 mmHg. 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 50 microM PPOH, afferent arteriolar diameter decreased by 29+/-4% when pressure was increased from 80-160 mmHg. Likewise, the sulphonimide derivative of PPOH, N-methylsulphonyl-6-(2-proparglyloxyphenyl) hexanamide (MS-PPOH, 50 microM), 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 25 microM DDMS, afferent arteriolar diameter decreased by 4+/-2% when pressure was increased from 80-160 mmHg. 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.

Amides↗

PubMed1999

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.

Adenosine Triphosphate↗