Intrinsic vascular dopamine - a key modulator of hypoxia-induced vasodilatation in splanchnic vessels.

Dopamine not only is a precursor of the catecholamines noradrenaline and adrenaline but also serves as an independent neurotransmitter and paracrine hormone. It plays an important role in the pathogenesis of hypertension and is a potent vasodilator in many mammalian systemic arteries, strongly suggesting an endogenous source of dopamine in the vascular wall. Here we demonstrated dopamine, noradrenaline and adrenaline in rat aorta and superior mesenteric arteries (SMA) by radioimmunoassay. Chemical sympathectomy with 6-hydroxydopamine showed a significant reduction of noradrenaline and adrenaline, while dopamine levels remained unaffected. Isolated endothelial cells were able to synthesize and release dopamine upon cAMP stimulation. Consistent with these data, mRNAs coding for catecholamine synthesizing enzymes, i.e. tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase, and dopamine-ß-hydroxylase were detected by RT-PCR in cultured endothelial cells from SMA. TH protein was detected by immunohistochemisty and Western blot. Exposure of endothelial cells to hypoxia (1% O2) increased TH mRNA. Vascular smooth muscle cells partially expressed catecholaminergic traits. A physiological role of endogenous vascular dopamine was shown in SMA, where D1 dopamine receptor blockade abrogated hypoxic vasodilatation. Experiments on SMA with endothelial denudation revealed a significant contribution of the endothelium, although subendothelial dopamine release dominated. From these results we conclude that endothelial cells and cells of the underlying vascular wall synthesize and release dopamine in an oxygen-regulated manner. In the splanchnic vasculature, this intrinsic non-neuronal dopamine is the dominating vasodilator released upon lowering of oxygen tension.

Modulation of cardiac ERG1 K(+) channels by cGMP signaling.

Different K(+) currents have been implicated in the myocardial action potential repolarization including the I(Kr). ERG1 alpha subunits, identified as the molecular correlate of I(Kr), have been shown to form heteromultimeric channels in the heart and their activity is modulated by a complex interplay of signal transduction events. Using electrophysiological techniques, we examined the effects of the cGMP-analogue 8-Br-cGMP on rat and guinea-pig papillary action potential duration (APD), on the biophysical properties of heterologously expressed homo- and heteromeric ERG1 channels, and on cardiac I(Kr). 8-Br-cGMP prolonged APD by about 25% after pharmacological inhibition of L-type Ca(2+) currents and I(Ks). The prolongation was completely abolished by prior application of the hERG channel blocker E-4031 or the protein kinase G (PKG) inhibitor Rp-8-Br-cGMPS. Expression analysis revealed the presence of both ERG1a and -1b subunits in rat papillary muscle. Both 8-Br-cGMP and ANP inhibited heterologously expressed ERG1b and even stronger ERG1a/1b channels, whereas ERG1a channels remained unaffected. The inhibitory 8-Br-cGMP effects were PKG-dependent and involved a profound ERG current reduction, which was also observed with cardiac AP clamp recordings. Measurements of I(Kr) from isolated mouse cardiomyocytes using Cs(+) as charge carrier exhibited faster deactivation kinetics in atrial than in ventricular myocytes consistent with a higher relative expression of ERG1b transcripts in atria than in ventricles. 8-Br-cGMP significantly reduced I(Kr) in atrial, but not in ventricular myocytes. These findings provide first evidence that through heteromeric assembly ERG1 channels become a critical target of cGMP-PKG signaling linking cGMP accumulation to cardiac I(Kr) modulation.

Erg K+ channels modulate contractile activity in the bovine epididymal duct.

The expression and functional role of ether-à-go-go-related gene (erg) K+ channels were examined in the bovine epididymal duct. Sperm transit through the epididymal duct relies on spontaneous phasic contractions (SC) of the peritubular smooth muscle wall. Isometric tension studies revealed SC-enhancing effects of the erg channel blockers E-4031, dofetilide, cisapride, and haloperidol and SC-suppressing effects of the activator NS-1643. In the corpus epididymidis, EC50 values of 32 nM and 8.3 microM were determined for E-4031 and NS-1643, respectively. E-4031 was also able to elicit contraction in epithelium-denuded corpus segments, which lacked SC. In the cauda region, E-4031 and NS-1643 exerted effects on agonist-induced contraction similar to those observed in the proximal duct. Experiments with nifedipine and thapsigargin suggested that the excitatory effects of E-4031 depended mainly on external calcium influx and not on intracellular calcium release. Western blot and RT-PCR assays revealed the expression of both, erg1a and erg1b, in all duct regions. Because erg1b appears to predominate in the epididymal duct, patch-clamp experiments were performed on heterologously expressed erg1b channels to investigate the sensitivity of this splice variant to NS-1643. In contrast to its effects on erg1a, NS-1643 induced a concentration-dependent current increase mainly due to a marked leftward shift in erg1b channel activation by approximately 30 mV at 10 microM, explaining the inhibitory effect of the drug on epididymal SC. In summary, these data provide strong evidence for a physiological role of erg1 channels in regulating epididymal motility patterns.