Norepinephrine (NE), a classic neurotransmitter in the sympathetic nervous system, induces vasoconstriction of canine isolated mesenteric vein that is accompanied by a sustained membrane depolarization. The mechanisms underlying the NE-elicited membrane depolarization remain undefined. In the present study we hypothesized that phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) are involved in the electrical field stimulation (EFS)-induced slow membrane depolarization (SMD) in canine isolated mesenteric vein. EFS (0.1–2 Hz, 0.1 ms, 15V, 10 s)-induced changes in the membrane potential were recorded with a conventional intracellular microelectrode technique and evaluated in the absence and presence of inhibitors of neuronal activity, α-adrenoceptors, membrane ion channels, PI3K, inositol 1,4,5-triphosphate (InsP3) receptors, and PKC. Activation of PI3Kγ and PKCζ in response to exogenous NE and clonidine in the absence and presence of receptor and kinase inhibitors were also determined.
Contractile responses to NE and clonidine (0.05 – 10 μM) were significantly diminished in the presence of yohimbine (0.1 μM). Exogenous NE (0.1 μM) and clonidine (1 μM) elicited SMD. The resting membrane potential of canine mesenteric vein smooth muscle cells was -68.8 ± 0.8 mV. EFS elicited a biphasic depolarization comprised of excitatory junction potentials and SMD that are purinergic and adrenergic in nature, respectively. The magnitude of the SMD in response to EFS at 0.5 Hz was 9.4 ± 0.7 mV. This response was reduced by 65–98% by the fast Na+ channel inhibitor tetrodotoxin (1 μM), by the inhibitor of N-type Ca2+ channels ω-conotoxin GVIA (5 nM), the non-selective α-adrenoceptor blocker phentolamine (1 μM), the selective α2-adrenoceptor blocker yohimbine (0.1 μM), the ion channel inhibitors niflumic acid (NFA, 100 μM), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 30 μM), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 200 μM), and Gd3+ (30 μM), and the PI3K inhibitors wortmannin (100 nM) and LY-294002 (10 μM). The SMD remained unchanged in the presence of the L-type Ca2+ channel blocker nicardipine (1 μM) and the InsP3 receptor blockers 2-aminoethoxydiphenylborate (2APB, 50 μM) and xestospongin C (3 μM). The inhibitor of PKC chelerythrine (1 μM), but not calphostin C (10 μM), diminished the SMD. Exogenous NE and clonidine (1 μM each) activated both PI3Kγ and PKCζ, and the activation of these kinases was abolished by preincubation of tissue with the α2-adrenoceptor blocker yohimbine.
Neuronally-released NE stimulates smooth muscle α2-adrenoceptors and activates PI3K and atypical PKC in the canine mesenteric vein. Events downstream of PKC lead to SMD and vasoconstriction. This represents a novel pathway for NE-induced membrane depolarization in a vascular smooth muscle preparation.