Impaired purinergic neurotransmission to mesenteric arteries in deoxycorticosterone acetate-salt hypertensive rats

Abstract

Sympathetic nerves release norepinephrine and ATP onto mesenteric arteries. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, there is increased arterial sympathetic neurotransmission attributable, in part, to impaired prejunctional regulation of norepinephrine release. Prejunctional regulation purinergic transmission in hypertension is less well understood. We hypothesized that alpha(2)-adrenergic receptor dysfunction alters purinergic neurotransmission to arteries in DOCA-salt hypertensive rats. Mesenteric artery preparations were maintained in vitro, and intracellular electrophysiological methods were used to record excitatory junction potentials (EJPs) from smooth muscle cells. EJP amplitude was reduced in smooth muscle cells from DOCA-salt (4+/-1 mV) compared with control arteries (9+/-1 mV; P<0.05). When using short trains of stimulation (0.5 Hz; 5 pulses), the alpha(2)adrenergic receptor antagonist yohimbine (1 micromol/L) potentiated EJPs in control more than in DOCA-salt arteries (180+/-35% versus 86+/-7%; P<0.05). Norepinephrine (0.1 to 3.0 micromol/L), the alpha(2)adrenergic receptor agonist UK 14304 (0.001 to 0.100 micromol/L), the A(1) adenosine receptor agonist cyclopentyladensosine (0.3 to 100.0 micromol/L), and the N-type calcium channel blocker omega-conotoxin GVIA (0.0003 to 0.1000 micromol/L) decreased EJP amplitude equally well in control and DOCA-salt arteries. Trains of stimuli (10 Hz) depleted ATP stores more completely, and the latency to EJP recovery was longer in DOCA-salt compared with control arteries. These data indicate that there is reduced purinergic input to mesenteric arteries of DOCA-salt rats because of decreased ATP bioavailability in sympathetic nerves. These data highlight the potential importance of impaired purinergic regulation of arterial tone as a target for drug treatment of hypertension.

Fig. 1. Electrophysiological properties of mesenteric arterial smooth muscle cells from DOCA-salt and control rats

A. Representative recordings showing EJPs are blocked by TTX (0.3 μM) or PPADS (10 μM). B. EJPs were blocked similarly by TTX and PPADS in control and DOCA-salt arteries (n=4; P > 0.05) indicating that they were neurogenic and mediated by activation of P2X receptors. C. Resting membrane potential (RMP) of mesenteric arterial smooth muscle cells (SMC) were depolarized in DOCA-salt compared to control tissues (−53 ± 2 mV vs. −60 ± 2 mV; n = 22; * = P < 0.05; DOCA-salt vs. control). EJPs in arteries from DOCA-salt rats were smaller than those in control arteries (4 ± 1 mV vs 8 ± 1 mV; n = 22; * = P < 0.05; DOCA-salt vs. control).

Fig. 2. Prejunctional α₂-AR and A₁-R, regulate ATP release from sympathetic nerves in control and DOCA-salt rats

Concentration response curves for A, UK 14,304 (n = 9), an α₂-AR agonist, B, norepinephrine (NE, n = 4) and C, cyclopentyladensosine (CPA, n=5), an A₁-adenosine receptor agonist were not different in tissues from control and DOCA-salt rats. Data points are mean ± SEM. Curves are non-linear fits of the Hill equation to the data points.

Fig. 3. N-type voltage-dependent calcium channel (VDCC) on peri-arterial sympathetic nerve fibers

A. Sympathetic nerve fibers were co-labeled with an anti-tyrosine hydroxylase (TH) antibody and an anti-α1B calcium channel (N-type calcium channel) subunit antibody. Immunoreactivity for TH and N-type VDCC (B) were observed in peri-arterial nerve fibers and nerve endings in the mesentery. C. Overlay of photomicrographs in A and B showing co-localization (yellow labeling) of TH and N-type VDCCs. Scale bar = 30 μM. D. CTX cause a concentration-dependent inhibition EJP amplitude (P > 0.05; control vs. DOCA-salt). Data points are mean ± SEM. Lines are non-linear fits of the Hill equation to the data points.

Fig. 4. EJP facilitation in arteries from control and DOCA-salt rats

EJPs were evoked using a short train (0.5 Hz, 10 s) of stimulation with or without yohimbine (0.1 μM). A. Representative traces show EJPs recorded from control and DOCA-salt treated arteries. B. Mean EJP amplitude in arteries from control and DOCA-salt rats without and with yohimbine (* = P < 0.05; control vs. control with yohimbine).

Fig. 5. EJP rundown caused by trains of stimulation in arteries from DOCA-salt and control rats

A train of stimulation (10 Hz) causes EJP facilitation and then rundown. A. Representative recordings of EJPs elicited by nerve stimulation for 5 seconds are shown for arteries from control (left) and DOCA-salt (right) rats. At this frequency of stimulation, individual EJPs summate to cause a sustained depolarization. EJPs rundown in arteries from DOCA-salt rats and the sustained depolarization declines to baseline level during the train of stimulation. B. Mean data from experiments similar to that shown in “A”. C. Summary data for EJP facilitation in DOCA-salt (gray bars) and control (black bars) arteries (* = P < 0.05; DOCA-salt vs. control). The ratio of peak EJP amplitude to the EJP amplitude at 30 s was calculated. This ratio was compared between control (black bars) and DOCA-salt animals (gray bars; * = P < 0.05).

Fig. 6. Impaired recovery of EJP amplitude after rundown in arteries from DOCA-salt rats

A single stimulus was given at 0.2, 0.5, 1 and 2 seconds after the end of a 10 Hz train (10 s). A. Representative recordings from a control artery ashowing complete recovery of EJP within 1s after the end of the 10 Hz train of stimulation. B. Summary data from control and DOCA-salt animals. In control arteries EJP amplitude at 0.2 s was no different from the initial EJP amplitude in the train. At 1 and 2 seconds after the end of the train, the EJP amplitudes were significantly greater than the initial EJP (* = P < 0.05; compared to initial amplitude). In DOCA-salt arteries the EJP at 0.2 s was significantly smaller than the initial EJP (* = P < 0.05; as compared to initial amplitude). EJP amplitudes during the recovery period in DOCA-salt arteries were significantly smaller than control arteries at 0.5, 1.0 and 2.0 s (^& = P < 0.05; DOCA-salt vs. control).