Pituitary adenylate cyclase-activating polypeptide (PACAP) potently dilates middle meningeal arteries: implications for migraine

Abstract

Migraine is a debilitating neurological disorder characterized by mild to severe headache that is often accompanied by aura and other neurological symptoms. Among proposed mechanisms, dilation of the dural vasculature especially the middle meningeal artery (MMA) has been implicated as one component underlying this disorder. Several regulatory peptides from trigeminal sensory and sphenopalatine postganglionic parasympathetic fibers innervating these vessels have been implicated in the process including pituitary adenylate cyclase-activating polypeptide (PACAP). Although PACAP has been well described as a potent dilator in many vascular beds, the effects of PACAP on the dural vasculature are unclear. In the current study, we examined the ability of PACAP to dilate MMAs that were isolated from rats and pressurized ex vivo. PACAP38 potently dilated pressurized MMAs with an EC(50) of 1 pM. The PAC1 receptor antagonist, PACAP(6-38), abolished MMA dilation caused by picomolar concentrations of PACAP. In contrast, cerebellar arteries isolated from the brain surface were ~1,000-fold less sensitive to PACAP than MMAs. Although cerebellar arteries expressed transcripts for all three PACAP receptor subtypes (PAC1, VPAC1, and VPAC2 receptors) by RT-PCR analyses, MMA demonstrated only PAC1 and VPAC2 receptor expression. Further, multiple variants of the PAC1 receptor were identified in the MMA. The expression of PAC1 receptors and the high potency of PACAP to induce MMA vasodilation are consistent with their potential roles in the etiology of migraine.

Fig. 1

Preparation of rat middle meningeal artery (MMA) for arteriograph studies. a Rat MMA embedded in the dura matter prior to dissection. Arrows indicate the MMA region typically used for ex vivo studies. b Isolated rat MMA cannulated on glass micropipettes mounted in an arteriograph chamber. Both ends of MMA were tied by suture thread, and the artery pressurized to 40 mmHg. Arterial diameter was measured using video edge detection and recorded using data acquisition software (details in “Methods”). One graduation of scale (e.g., 0 to 1) represents 100 μm

Fig. 2

Pressure-induced myogenic tone in isolated rat middle meningeal arteries (MMAs). a Diameter measurements of isolated rat MMA obtained in response to stepwise increases in intravascular pressure from 10 to 120 mmHg. Squares with solid line represent active diameter measurements obtained in artificial cerebral spinal fluid (aCSF) (n=4). Circles with dashed line represent maximally dilated (passive) diameter measurements obtained from the same arteries in Ca²⁺-free aCSF containing the vasodilators diltiazem (100 μM) and forskolin (1 μM). b Pressure-induced constriction (myogenic tone) in rat MMAs superfused with aCSF ex vivo. Constriction is expressed as a percent decrease from maximally dilated diameter obtained at the same intravascular pressure (n=4)

Fig. 3

Low picomolar concentrations of PACAP, but not VIP, dilate isolated pressurized rat middle meningeal arteries (MMAs). a Cumulative concentration response curves of PACAP and VIP obtained from rat MMAs pressurized to 40 mmHg ex vivo. Arteries were exposed to aCSF containing each concentration of PACAP38 or VIP for 20 min. Dilation to PACAP38 or VIP are expressed as percentage of maximum dilation obtained in the presence of Ca²⁺-free aCSF containing 100 μM diltiazem and 1 μM forskolin. p<0.05 by unpaired t test, n=4. b, c Diameter recordings of an isolated MMA treated with aCSF containing a single concentration (3 pM) of PACAP38 (b) and VIP (c). At the end of the experiment, maximally dilated diameter was obtained in the presence of Ca²⁺-free aCSF containing 100 μM diltiazem and 1 μM forskolin (shown as passive)

Fig. 4

Block of picomolar PACAP-induced dilation of isolated pressurized middle meningeal artery (MMA) by the PAC1 receptor antagonist PACAP(6-38). a–d Diameter recordings from isolated MMAs obtained in the absence (a, c) and presence (b, d) of the PAC1 antagonist, PACAP(6-38). Arteries, pressurized to 40 mmHg, were treated with either 3 pM PACAP38 (a, b) or 3 nM PACAP38 (c, d). Maximum dilation (passive) was obtained by treatment with 100 μM diltiazem and 1 μM forskolin in Ca²⁺-free aCSF. e Summary data of PACAP38-induced dilation in the absence or presence of PACAP(6-38). PACAP(6-38) abolished 3 pM PACAP38-induced vasodilation but caused only partial inhibition of 3 nM PACAP38-induced vasodilation in rat MMAs (n=4 each). Diameter changes by PACAP38 treatment were expressed as percentage of maximum dilation. *p<0.05 vs PACAP-induced vasodilation in the absence of PACAP(6-38)

Fig. 5

Nanomolar, but not picomolar, concentrations of PACAP38 and VIP dilate pressurized cerebellar arteries. a Diameter recording obtained from pressurized rat cerebellar artery treated with PACAP38. PACAP38 induced dilation at 3 nM but not 3 pM. Maximum dilation was obtained using a combination of 100 μM diltiazem and 1 μM forskolin in Ca²⁺-free aCSF (passive). b Diameter recording obtained from pressurized rat cerebellar artery treated with VIP. VIP-induced dilation at 3 nM but not 3 pM. Maximum dilation was obtained using a combination of 100 μM diltiazem and 1 μM forskolin in Ca²⁺-free aCSF (passive). c Summarized data of PACAP38 and VIP treatment in rat cerebellar arteries. PACAP38-induced diameter changes are expressed as percentage of maximum dilation (n=4 each). *p<0.05 by paired t test

Fig. 6

PACAP receptor transcripts are differentially expressed in rat middle meningeal and cerebellar arteries. Total RNA from rat MMA and cerebellar arteries were reverse-transcribed for semiquantitative PCR analyses of PAC1 (413 bp), VPAC1 (323 bp), and VPAC2 (396 bp) receptor transcript expression. a Unlike cerebellar artery preparations which demonstrated mRNA for all three receptor subtypes (right panel, n=5), the MMA appeared to express only PAC1 and VPAC2 receptor transcripts (left panel, n=5). b To examine whether there were differences in PAC1 receptor isoform expression levels between the two arteries, primers flanking the third cytoplasmic loop were employed in the PCR studies. While cerebellar arteries appeared to possess predominantly the PAC1null receptor isoform (right panel, neither Hip nor Hop; 303 bp), the MMA expressed near-equal levels of the PAC1null (303 bp) and the one-cassette PAC1 receptor variant (387 bp; left panel). Each lane represents an independent sample replicate. Amplification of the same templates for GAPDH demonstrates approximate equal sample input. c The specific one-cassette PAC1 receptor variant could be identified by diagnostic restriction digest; isolation of the MMA 387-bp PAC1 receptor band followed by Blp I restriction enzyme incubation resulted in amplicon digestion to a smaller 292-bp product diagnostic of the Hop receptor variant