PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries

Abstract

Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction.

Keywords: Bayliss; calcium; diacylglycerol; phospholipase C.

Fig. 1.

Effects of phospholipase C inhibitors on myogenic tone of isobaric murine mesenteric arteries. The myogenic response of pressurized murine mesenteric arteries under control and in the presence of U-73122 (3 μM, n = 8; A), edelfosine (20 μM, n = 7, B), and D609 (10 μM, n = 5, C) is shown. Passive curves are obtained in 0 Ca²⁺ solution (*P < 0.05, active vs. drug).

Fig. 2.

Effects of phospholipase C (PLC) inhibitors on the contractile response of isobaric murine mesenteric arteries to stimulation with KCl and phenylephrine (PE). A: U-73122 (3 μM, n = 5, *P < 0.05) wide-spectrum PLC inhibitor. B: edelfosine (20 μM, n = 6, P = 0.06) phosphoinositide-specific (PI-PLC) inhibitor. C: D609 [10 μM (n = 7; KCl, n = 4 PE), *P < 0.05] phosphatidylcholine-specific (PC-PLC)/smooth muscle (SM) synthase inhibitor. Arteries were stimulated with 60 mM KCl or 10 μM PE at 70 mmHg, 37°C.

Fig. 3.

Ca²⁺ signals in pressurized murine mesenteric arteries before and after development of myogenic tone. A: the diameter of a pressurized (70 mmHg) mesenteric artery is shown. Ca²⁺ fluorescence signals were obtained before (i, arrow) and after (ii, arrow) development of myogenic tone as shown in the diameter trace. B: typical x–y confocal scans (first column) used to calculate fluorescence values from single arterial myocytes show a virtual absence of high-amplitude, long-duration Ca²⁺ waves after development of myogenic tone (Bii, bottom, fluo 2-AM). C: summary bar graph depicting the frequency of Ca²⁺ waves in pressurized arterial myocytes (n = 4, *P < 0.05) before and after development of myogenic tone.

Fig. 4.

Frequency of Ca²⁺ sparks in isobaric murine mesenteric artery smooth muscle cells during maintained myogenic constriction. A and B: representative line scans of pressurized murine mesenteric arteries loaded with Ca²⁺ indicator illustrate the prevalence of Ca²⁺ sparks before and after the development of myogenic tone. The frequency of sparks is calculated for a 100-μm-wide section of a line scan, thus yielding the unit spark·100 μm⁻¹·s⁻¹. C: summary bar graph indicating a decrease in the frequency of Ca²⁺ sparks in smooth muscle cells during established myogenic tone. A and B, bottom: fluorescence units (F); bar is equivalent to 800 units. *P < 0.05, no tone vs. myogenic tone.

Fig. 5.

Modulation of myogenic tone by sarcoplasmic reticulum Ca²⁺ stores. The effects of blockade of ryanodine receptors (30 μM ryanodine) (A) and Ca²⁺-dependent K⁺ (K_Ca) channels [100 nM iberiotoxin (IbTx)] (B) on myogenic tone of pressurized murine mesenteric arteries were measured. C: xestospongin C (5 μM, XeC) was used to block Ca²⁺ release via inositol 1,4,5-trisphosphate (IP₃) receptors. XeC did not affect myogenic tone but eliminated the contractile response to bath-applied PE (10 μM). D: incubation of arteries with 10 μM cyclopiazonic acid (CPA) significantly increased myogenic tone. Passive diameter was measured with 0 Ca²⁺ perfusate (*P < 0.01). ctr, Control.

Fig. 6.

Effects of inhibition of PC-PLC/sphingomyelin synthase on pressure-induced Ca²⁺ response of murine mesenteric smooth muscle cells. A: the Förster resonance energy transfer (FRET) ratio response in a pressurized mesenteric artery expressing exMLCK biosensor is shown under control and after treatment with D609 (10 μM, n = 3). B: summary of the pressure-induced FRET response of mesenteric arteries [normalized ratio at a particular pressure (R) to that at 30 mmHg (R₃₀)] before and after treatment with D609, n = 3. C: myogenic tone in mesenteric arteries under control and after treatment with SKF-96365, n = 3. *P < 0.05, control vs. SKF-96365. D: FRET response of exMLCK biosensor to depolarization with 117 mM K⁺ in the absence and presence of D609 (n = 3).

Fig. 7.

Effects of membrane-permeable diacylglycerol (DAG) analog on diameter of pressurized murine mesenteric arteries. A: the diameter response of a pressurized mesenteric artery in response to treatment with 1,2-dioctanoyl-sn-glycerol (DOG, 20 μM, 50 mmHg) is shown. B: summary of the response of arteries to treatment with DOG (n = 5).

Fig. 8.

Proposed role of PC-PLC/sphingomyelin synthase in the myogenic response of murine mesenteric arteries. Mechanical stretch of the plasma membrane leads to ligand-independent activation of G protein-coupled receptors [GPCRs, including angiotensin type 1 (AT₁)]. D609-sensitive PC-PLC and/or sphingomyelin synthase activate and use phosphatidylcholine to yield DAG metabolite. DAG activates plasma membrane currents, including transient receptor potential C (TRPC) family members and protein kinase C (PKC). Inhibition of a K⁺ current also contributes to depolarization. The resultant increase in Ca²⁺ influx and Ca²⁺ sensitivity thus leads to myogenic vasoconstriction.