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. 2018 Feb;222(2):10.1111/apha.12913.
doi: 10.1111/apha.12913. Epub 2017 Jul 13.

Mechanisms of sphingosine-1-phosphate-mediated Vasoconstriction of Rat Afferent Arterioles

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Free PMC article

Mechanisms of sphingosine-1-phosphate-mediated Vasoconstriction of Rat Afferent Arterioles

Z Guan et al. Acta Physiol (Oxf). .
Free PMC article

Abstract

Aim: Sphingosine-1-phosphate (S1P) influences resistance vessel function and is implicated in renal pathological processes. Previous studies revealed that S1P evoked potent vasoconstriction of the pre-glomerular microvasculature, but the underlying mechanisms remain incompletely defined. We postulated that S1P-mediated pre-glomerular microvascular vasoconstriction involves activation of voltage-dependent L-type calcium channels (L-VDCC) and the rho/rho kinase pathway.

Methods: Afferent arteriolar reactivity was assessed in vitro using the blood-perfused rat juxtamedullary nephron preparation, and diameter was measured during exposure to physiological and pharmacological agents.

Results: Exogenous S1P (10-9 -10-5 mol L-1 ) evoked concentration-dependent vasoconstriction of afferent arterioles. Superfusion with nifedipine, a L-VDCC blocker, increased arteriolar diameter by 39 ± 18% of baseline and significantly attenuated the S1P-induced vasoconstriction. Superfusion with the rho kinase inhibitor, Y-27632, increased diameter by 60 ± 12% of baseline and also significantly blunted vasoconstriction by S1P. Combined nifedipine and Y-27632 treatment significantly inhibited S1P-induced vasoconstriction over the entire concentration range tested. In contrast, depletion of intracellular Ca2+ stores with the Ca2+ -ATPase inhibitors, thapsigargin or cyclopiazonic acid, did not alter the S1P-mediated vasoconstrictor profile. Scavenging reactive oxygen species (ROS) or inhibition of nicotinamide adenine dinucleotide phosphate oxidase activity significantly attenuated S1P-mediated vasoconstriction.

Conclusion: Exogenous S1P elicits potent vasoconstriction of rat afferent arterioles. These data also demonstrate that S1P-mediated pre-glomerular vasoconstriction involves activation of L-VDCC, the rho/rho kinase pathway and ROS. Mobilization of Ca2+ from intracellular stores is not required for S1P-mediated vasoconstriction. These studies reveal a potential role for S1P in the modulation of renal microvascular tone.

Keywords: Ca2+ signalling; cyclopiazonic acid; reactive oxygen species; renal microvascular reactivity; tempol; thapsigargin.

Figures

Figure 1
Figure 1. Effect of pharmacological agents on baseline diameters of afferent arterioles
(a): afferent arteriolar diameters prior to exposure to pharmacological agents (baseline, white columns) and after exposure to pharmacological agent(s) but before administration of S1P (blue columns). (b): the same data normalized as a percentage of the baseline diameter for each group. Con, control group; Nif, nifedipine group; Tg, thapsigargin; CPA, cyclopiazonic acid; Y, Y-27632; Nif+Y, nifedipine+Y-27632; Temp, tempol; Apo, apocynin. Values are expressed as mean ± SEM. *P < 0.05 vs. baseline in the same group.
Figure 1
Figure 1. Effect of pharmacological agents on baseline diameters of afferent arterioles
(a): afferent arteriolar diameters prior to exposure to pharmacological agents (baseline, white columns) and after exposure to pharmacological agent(s) but before administration of S1P (blue columns). (b): the same data normalized as a percentage of the baseline diameter for each group. Con, control group; Nif, nifedipine group; Tg, thapsigargin; CPA, cyclopiazonic acid; Y, Y-27632; Nif+Y, nifedipine+Y-27632; Temp, tempol; Apo, apocynin. Values are expressed as mean ± SEM. *P < 0.05 vs. baseline in the same group.
Figure 2
Figure 2. Effect of nifedipine on S1P–induced afferent arteriolar vasoconstriction
(a): afferent arteriolar responses to increasing concentrations of S1P in the absence (diamonds) and presence of the voltage-dependent L-type calcium channel blocker, nifedipine (10−6 mol L−1, squares). Data represent the actual diameters from each group. Con, control diameters averaged from the last 5-min incubation period in the absence or presence of nifedipine but prior to administration of S1P. (b): the same data normalized as a percentage of the control diameter for each group. Values are expressed as mean ± SEM. *P < 0.05 vs. control diameter in the same group. †P < 0.05 vs. vasoconstriction with S1P alone at the same concentration.
Figure 3
Figure 3. Effect of thapsigargin on S1P–induced afferent arteriolar vasoconstriction
(a): afferent arteriolar responses to increasing concentrations of S1P in the absence (diamonds) and presence of the Ca2+-ATPase inhibitor, thapsigargin (10−6 mol L−1, squares). Data represent the actual diameters from each group. The S1P alone data are taken from Figure 2. Con, control diameters averaged from the last 5-min incubation period in the absence or presence of thapsigargin but prior to administration of S1P. (b): the same data normalized as a percentage of the control diameter for each group. Values are expressed as mean ± SEM. *P < 0.05 vs. control diameter in the same group.
Figure 4
Figure 4. Effect of cyclopiazonic acid on S1P–induced afferent arteriolar vasoconstriction
(a): afferent arteriolar responses to increasing concentrations of S1P in the absence (diamonds) and presence of the selective Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA, 10−4 mol L−1, squares). Data represent the actual diameters from each group. The S1P alone data are taken from Figure 2. Con, control diameters averaged from the last 5-min incubation period in the absence or presence of CPA but prior to administration of S1P. (b): the same data normalized as a percentage of the control diameter for each group. Values are expressed as mean ± SEM. *P < 0.05 vs. control diameter in the same group.
Figure 5
Figure 5. Effect of the rho kinase inhibitor, Y-27632 on S1P–induced afferent arteriolar vasoconstriction
(a): afferent arteriolar responses to increasing concentrations of S1P in the absence (diamonds) and presence of the rho kinase inhibitor, Y-27632 (10−5 mol L−1, squares). Data represent the actual diameters from each group. The S1P alone data are taken from Figure 2. Con, control diameters averaged from the last 5-min incubation period in the absence or presence of Y-27632 but prior to administration of S1P. (b): the same data normalized as a percentage of the control diameter for each group. Values are expressed as mean ± SEM. *P < 0.05 vs. control diameter in the same group. †P < 0.05 vs. vasoconstriction with S1P alone at the same concentration.
Figure 6
Figure 6. Effect of a combination of nifedipine and Y-27632 on S1P–induced afferent arteriolar vasoconstriction
(a): afferent arteriolar responses to increasing concentrations of S1P in the absence (diamonds) and presence of both nifedipine (10−6 mol L−1) and Y-27632 (10−5 mol L−1, squares) combined. Data represent the actual diameters from each group. The S1P alone data are taken from Figure 2. Con, control diameters averaged from the last 5-min incubation period in the absence or presence of the combination of nifedipine and Y-27632 but prior to administration of S1P. (b): the same data normalized as a percentage of the control diameter for each group. Values are expressed as mean ± SEM. *P < 0.05 vs. control diameter in the same group. †P < 0.05 vs. vasoconstriction with S1P alone at the same concentration.
Figure 7
Figure 7. Effects of tempol, a reactive oxygen species scavenger and apocynin, the NADPH oxidase inhibitor on S1P–induced afferent arteriolar vasoconstriction
(a): afferent arteriolar responses to increasing concentrations of S1P in the absence (diamonds) and presence of tempol (10−3 mol L−1, squares) or apocynin (10−4 mol L−1, triangles). Data represent the actual diameters from each group. The S1P alone data are taken from Figure 2. Con, control diameters averaged from the last 5-min incubation period in the absence or presence of tempol or apocynin but prior to administration of S1P. (b): the same data normalized as a percentage of the control diameter for each group. Values are expressed as mean ± SEM. *P < 0.05 vs. control diameter in the same group. †P < 0.05 vs. vasoconstriction with S1P alone at the same concentration.

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