α1-Adrenergic receptor subtype function in fetal and adult cerebral arteries

In the developing fetus, cerebral artery (CA) contractility demonstrates significant functional differences from that of the adult. This may be a consequence of differential activities of α1-adrenergic receptor (α1-AR) subtypes. Thus we tested the hypothesis that maturational differences in adrenergic-mediated CA contractility are, in part, a consequence of differential expression and/or activities of α1-AR subtypes. In CA from fetal (∼140 days) and nonpregnant adult sheep, we used wire myography and imaging, with simultaneous measurement of tension and intracellular Ca2+ concentration ([Ca2+]i), radioimmunoassay, and Western immunoblots to examine phenylephrine (Phe)-induced contractile responses. The α1A-AR antagonists (5-MU and WB-4101) completely inhibited Phe-induced contraction in adult but not fetal CA; however, [Ca2+]i increase was reduced significantly in both age groups. The α1D-AR antagonist (BMY-7378) blocked both Phe-induced contractions and Ca2+ responses to a significantly greater extent in adult compared with fetal CA. In both age groups, inhibition of α1A-AR and α1B-AR, but not α1D-AR, significantly reduced inositol 1,4,5-trisphosphate responses to Phe. Western immunoblots demonstrated that the α1-AR subtype expression was only ∼20% in fetal CA compared with the adult. Moreover, in fetal CA, the α1D-AR was expressed significantly greater than the other two subtypes. Also, in fetal but not adult CA, Phe induced a significant increase in activated ERK1/2; this increase in phosphorylated ERK was blocked by α1B-AR (CEC) and α1D-AR (BMY-7378) inhibitors, but not by α1A-AR inhibitors (5-MU or WB-4101). In conclusion, in the fetal CA, α1B-AR and α1D-AR subtypes play a key role in contractile response as well as in ERK activation. We speculate that in fetal CA α1B-AR and α1D-AR subtypes may be a critical factor associated with cerebrovascular growth and function.

Maintenance of gasping and restoration of eupnea after hypoxia is impaired following blockers of α1-adrenergic receptors and serotonin 5-HT2 receptors

In severe hypoxia or ischemia, normal eupneic breathing fails and is replaced by gasping. Gasping serves as part of a process of autoresuscitation by which eupnea is reestablished. Medullary neurons, having a burster, pacemaker discharge, underlie gasping. Conductance through persistent sodium channels is essential for the burster discharge. This conductance is modulated by norepinephrine, acting on α1-adrenergic receptors, and serotonin, acting on 5-HT2 receptors. We hypothesized that blockers of 5-HT2 receptors and α1-adrenergic receptors would alter autoresuscitation. The in situ perfused preparation of the juvenile rat was used. Integrated phrenic discharge was switched from an incrementing pattern, akin to eupnea, to the decrementing pattern comparable to gasping in hypoxic hypercapnia. With a restoration of hyperoxic normocapnia, rhythmic, incrementing phrenic discharge returned within 10 s in most preparations. Following addition of blockers of α1-adrenergic receptors (WB-4101, 0.0625–0.500 μM) and/or blockers of 5-HT2 (ketanserin, 1.25–10 μM) or multiple 5-HT receptors (methysergide, 3.0–10 μM) to the perfusate, incrementing phrenic discharge continued. Fictive gasping was still induced, although it ceased after significantly fewer decrementing bursts than in preparations than received no blockers. Moreover, the time for recovery of rhythmic activity was significantly prolonged. This prolongation was in excess of 100 s in all preparations that received both WB-4101 (above 0.125 μM) and methysergide (above 2.5 μM). We conclude that activation of adrenergic and 5-HT2 receptors is important to sustain gasping and to restore rhythmic respiratory activity after hypoxia-induced depression.

Opposing effects of α1-adrenergic receptor subtypes on Ca2+ and pH homeostasis in rat cardiac myocytes

We examined the effect of α1-adrenergic receptor (AR) subtypes on contraction, cytosolic Ca2+ concentration ([Ca2+]i), and cytosolic pH (pHi) of rat ventricular myocytes loaded with the Ca2+ indicator indo 1 or the pH indicator carboxy-seminaphthorhodafluor-1. Nonselective α1-AR stimulation was effected with phenylephrine plus nadolol. α1-AR subtype stimulation was achieved with α1-AR and chloroethylclonidine (CEC) or with α1-AR and WB-4101. Cells were in bicarbonate buffer with 0.5 mM Ca2+ and were electrically stimulated at 0.5 Hz. Results show that 1) nonselective α1-AR stimulation increased twitch and [Ca2+]itransient amplitudes, myofilament response to Ca2+, and pHi; 2) α1-AR plus CEC increased twitch and [Ca2+]itransient amplitudes and also enhanced myofilament response to Ca2+ via cytosolic alkalinization; 3) α1-AR plus WB-4101 decreased twitch and [Ca2+]itransient amplitudes and also pHi; and 4) cytosolic acidification due to α1-AR plus WB-4101 was abolished by protein kinase C inhibition (staurosporine pretreatment) or downregulation (prolonged exposure to phorbol esters). In summary, the net effects of α1-adrenergic stimulation on contraction, [Ca2+]i, and pHi are due to opposing WB-4101- and CEC-sensitive α1-AR subtype signaling pathways.

Alpha 1-adrenoceptor subtypes mediating inotropic and electrophysiological effects in mammalian myocardium

Stimulation of alpha 1-adrenoceptors produces a positive inotropic effect in rat and rabbit ventricular myocardium via different mechanisms, the prolongation of action potential duration (APD) exclusively in the former and an increase in myofibrillar Ca2+ sensitivity in large part in the latter. This study was designed to determine whether the two inotropic mechanisms are mediated by different alpha 1-adrenoceptor subtypes. In rat papillary muscles, the positive inotropic effect and APD prolongation induced by phenylephrine (in the presence of propranolol) were inhibited by WB-4101, but not affected by chlorethylclonidine (CEC). WB-4101, but not CEC, blocked the phenylephrine-induced inhibition of the transient outward current (Ito) in rat ventricular cells. On the other hand, WB-4101 and CEC each antagonized the positive inotropic effect of phenylephrine in rabbit papillary muscles. However, the phenylephrine-induced APD prolongation observed in rabbit papillary muscles was blocked only by WB-4101. These results indicate that the WB-4101 sensitive alpha 1-adrenoceptor subtype mediates the positive inotropism that is correlated with the APD prolongation resulting from Ito reduction, whereas the CEC-sensitive subtype mediates the positive inotropism that is probably associated with increased myofibrillar Ca2+ sensitivity. Radioligand binding studies with [3H] prazosin showed a similar ratio of alpha 1A-to alpha 1B-adrenoceptor subtypes in rat and rabbit ventricular myocardium, implying that the different degree of contribution of each action mechanism to the overall inotropic effect in the two species cannot be explained by distribution of the alpha 1-adrenoceptor subtypes.

Refractory period response of cardiac Purkinje tissue to alpha 1- and alpha 2-adrenergic influences

Our purpose was to characterize Purkinje responses in vivo to alpha 1- and alpha 2-adrenergic stimulation in sinoaortically denervated and vagotomized dogs pretreated with metoprolol (1 mg/kg). We measured Purkinje relative refractory period (PRRP) responses to norepinephrine (NE) and phenylephrine (PE) with prazosin and/or yohimbine, WB-4101, and chloralethylclonidine (CEC) in varying doses. Results were as follows: PE infusion (25 micrograms.kg-1.min-1) prolonged PRRP (9.6 +/- 1.4 ms; a 4.1 +/- 0.4% change). Prazosin blocked PRRP prolongation with PE at 7 x 10(-8) M/kg (P < 0.05). Yohimbine did not attenuate PRRP prolongation with PE either alone or in combination with prazosin. NE infusion (0.8 micrograms.kg-1.min-1) also prolonged PRRP (9.2 +/- 2.3 ms; a 4.8 +/- 1.0% change). In contrast neither prazosin nor yohimbine at any dose (up to 10(-6) M/kg) totally blocked the prolongation with NE infusion. However, with prazosin (2 x 10(-7) M/kg) pretreatment, yohimbine blocked PRRP prolongation, significant at 7 x 10(-8) M/kg (P < 0.05). In separate experiments with yohimbine pretreatment at 7 x 10(-8) M/kg, PRRP prolongation with either PE or NE infusion was blocked equipotently with WB-4101 and CEC at 7 x 10(-8) M/kg. However, CEC did not block mean arterial pressure (MAP) responses to PE or NE infusion unlike WB-4101. We concluded that both subclasses of alpha 1-adrenergic antagonists equipotently block PRRP prolongation by alpha-agonists despite different effects on MAP. Purkinje refractoriness is also prolonged by alpha 2-adrenergic stimulation acting at the cell membrane.